Natural killer cell lines and methods of use

ABSTRACT

This invention relates to a natural killer cell line termed NK-92 and to NK-92 cell lines that have been modified by transfection with a vector to confer advantageous properties. Additionally, the invention provides an NK-92 cell, an NK-92 cell modified by transfection with a vector conferring advantageous properties, which is unable to proliferate and which preserves the effective cytotoxic activity. The invention provides a modified NK-92 cell line that is transfected with a vector encoding a cytokine that promotes the growth of NK-92 cells. In a significant embodiment, the cytokine is interleukin 2. The invention additionally provides a modified NK-92 cell line that is transfected with a vector that expresses a thymidine kinase gene. The invention further provides a modified NK-92 cell line that is transfected with a vector that expresses a β 2  microglobulin that has lost the ability to bind to T-cell receptors.

RELATED APPLICATION

This is a continuation-in-part of U.S. patent application Ser. No.09/403,910 filed Oct. 27, 1999 (now abandoned), which was a nationalizedapplication of PCT Application No. PCT/US98/08672 filed Apr. 30, 1998,which claims priority to U.S. Provisional Patent Application No.60/045,885 filed Apr. 30, 1997.

FIELD OF THE INVENTION

This invention relates to natural killer cells and their use in thetreatment of pathologies related to cancer or viral infections.Specifically, a particular cell line, NK-92, and modifications thereof,are disclosed. These cells are shown to be highly effective in thetreatment of these pathologies.

BACKGROUND OF THE INVENTION

Certain cells of the immune system have cytotoxic activity againstparticular target cells. Cytotoxic T lymphocytes (CTLs) are specificallydirected to their targets via antigen-derived peptides bound to MHCclass !-specific markers. Natural killer (NK) cells, however, are not sorestricted. NK cells, generally representing about 10-15% of circulatinglymphocytes, bind and kill target cells, including virus-infected cellsand many malignant cells, nonspecifically with regard to antigen andwithout prior immune sensitization (Herberman et al., Science 214.24(1981)). Killing of target cells occurs by inducing cell lysis. MHCclass restriction likewise is not involved. In these ways the activityof NK cells differs from antigen-specific and MHC class-specific Tcells, such as cytotoxic T lymphocytes. Use of NK cells in theimmunotherapy of tumors and malignancies is suggested by theseproperties, since many tumors are MHC class I deficient and therefore donot attract CTL activity. Adhesion molecules may also be involved in thetargeting of NK cells; for example, it is observed that the Fcγ receptor(CD16) is expressed on NK cells. NK cells are large granular lymphocyteswhich lack CD3, and in addition to CDI6, also may express Leu19 (Lanieret al., J. Immunol. 136; 4480 (1986)).

NK cells are activated when exposed to cytokines such as interleukin-2(IL-2), IL-7, IL-12, and interferons (Alderson et al., J. Exp. Med.172:577-587 (1990); Robertson et al., J. Exp. Med. 175:779-788 (1992)).The resulting cells are called lymphokine activated killer (LAK) cells.The spectrum of target cells is altered in activated NK cells comparedto nonactivated cells, although the mechanism of killing may beidentical or similar (Philips et al., J. Exp. Med. 164:814-825 (1986)).

More generally, killing activity in the cells of the immune system maybe induced by treating a population of cells, such as peripheral bloodmononuclear cells (PBMCs), with lymphokines. Such preparations containLAK cells. LAK cells may also be generated from autologous samples ofperipheral blood lymphocytes. LAK cells have antitumor killing activitywhile having essentially no effect on normal cells. They appear to purgeleukemia (Long et al., Transplantation 46:433 (1988); Xhou et al., Proc.Am. Assoc. Cancer Res. 34:469 (1993; abstract)), lymphoma (Schmidt-Wolfet al., J. Exp. Med. 174: 139 (1991); Gambacorti-Passerini et al., Br.J. Haematol. 18:197 (1991)) and neuroblastoma (Ades et al., Clin.Immunol. Immunopathol. 46:150 (1988)). NK cells, activated NK cells, andLAK cells are distinguishable by their cell surface markers and by theidentity of the target cells that they kill.

Activated and expanded (i.e., cultured to proliferate) NK cells and LAKcells have been used in both ex vivo therapy and in vivo treatment inpatients with advanced cancer. Some success with ex vivo therapy hasbeen observed in bone marrow related diseases, such as leukemia, breastcancer and certain types of lymphoma. In vivo treatment may be directedtoward these and other forms of cancer, including malignant melanoma andkidney cancer (Rosenberg et al., N. Engl. J. Med. 316:889-897 (1987)).LAK cell treatment requires that the patient first receive IL-2,followed by leukophoresis and then an ex vivo incubation and culture ofthe harvested autologous blood cells in the presence of IL-2 for a fewdays. The LAK cells must be reinfused along with relatively high dosesof IL-2 to complete the therapy. This purging treatment is expensive andcan cause serious side effects. These include fluid retention, pulmonaryedema, drop in blood pressure, and high fever. In some cases in whichthese side effects occur, intensive care unit management is required.

Purging techniques have been applied in other circumstances as well.Cytotoxic drugs or monoclonal antibodies combined with complement, andtoxins, may be administered in order to bring about remission. In suchcases bone marrow or blood stem cells, purged to reduce the number ofresidual leukemic cells present, have been infused back into the patientafter the drug treatment (Uckun et al., Blood 79:1094 (1992)). Genemarking studies have shown that unpurged bone marrow may contribute torelapse in patients presumed to be in remission (Brenner et al., Lancet341:85 (1993)). This suggests that some form of purging of autologousmarrow or blood prior to transplantation is necessary (Klingemann etal., Biol. Blood Marrow Transplant. 2:68-69 (1996)).

Recently, preclinical studies have also demonstrated promising antitumoractivity in vivo with a lethally irradiated, MHC-unrestricted, cytotoxicT-cell leukemic clone (TALL-104) (Cesano et al., Cancer Immunol.Immunofher. 40:139-151 (1995); Cesano et al., Blood 87:393-403 (1996)).These cells were derived from leukemia T cell lines obtained frompatients having acute T lymphoblastic leukemias (ALL). They bear the CD3cell surface marker, but not the CD56 marker, in distinction to NK cellswhich have the converse immunophenotype (CD3⁻ CD56⁺). Adoptive transferof these cells was able to eliminate human leukemic cell lines inxenografted severe combined immunodeficient (SCID) mice and to induceremissions of spontaneous lymphomas in dogs without producing T-cellleukemia in the animal models (Cesano et al. (1995); Cesano et al.(1996); Cesano et al., J. Clin. Invest. 94:1076-1084 (1994); Cesano etal., Cancer Res. 56:3021-3029 (1996)).

In spite of the advantageous properties of NK cells in killing tumorcells and virus-infected cells, they remain difficult to work with andto apply in immunotherapy. It is difficult to expand NK cells ex vivothat maintain their tumor-targeting, tumoricidal, and viricidalcapabilities in vivo. This remains a major obstacle to their clinicaluse in adoptive cell immunotherapy (Melder et al., Cancer Research48:3461-3469 (1988); Stephen et al., Leuk. Lymphoma 377-399 (1992);Rosenberg et al., New Engl. J. Med. 316:889-897 (1987)). Studies of themechanisms whereby NK cells exert their tumoricidal and viricidaleffects are also limited by difficulties in enriching the NK cellfractions without compromising their biological functions and inobtaining pure NK cells that are not contaminated by T cells or otherimmune effector cells. In an attempt to overcome these problems, manyinvestigators have turned to the use of established NK-like cell linesto explore the mechanisms whereby target cells are susceptible tocytotoxic cells (Hercend et al., Nature 301:158-160 (1983); Yodoi etal., J. Immunol. 134:1623-1630 (1985); Fernandez et al., Blood67:925-930 (1986); Robertson et al., Exp. Hematol. 24:406-415 (1996);Gong et al., Leukemia 8:652-658 (1994)). NK cell lines described inearlier work carry T lymphocyte-associated surface markers, in spite ofthe fact that they were developed from precursor cells depleted of Tcells (Rosenberg, et al. (1987); Hercend, et al., (1983)).

There thus remains a need for a method of treating a pathology relatedto cancer or a viral infection with a natural killer cell line thatmaintains viability and therapeutic effectiveness against a variety oftumor classes. This need encompasses therapeutic methods in whichsamples from a mammal are treated ex vivo with natural killer cells, aswell as methods of treatment of these pathologies with natural killercells in vivo in a mammal. There is also a need for a natural killercell line that maintains its own propensity for viability and cytolyticactivity by secreting a cytokine which fosters these properties. Therealso remains a need for such natural killer cell lines which aremodified to be more effective, convenient, and/or useful in treatment ofcancer and viral infection. It is the objective of this invention toprovide NK cells and methods that address these needs.

SUMMARY OF THE INVENTION

The cell line described by Gong et al. (1994), termed NK-92,proliferates in the presence of IL-2 and has high cytolytic activityagainst a variety of cancers. The present invention employs the NK-92cell line, as well as modified NK-92 cell lines, to provide cancertreatment and virus treatment systems. The invention also provides thevectors that transfect NK-92, as well as the modified NK-92 cells. Forpurposes of this invention and unless indicated otherwise, the term“NK-92” is intended to refer to the original NK-92 cell lines as well asthe modified NK-92 cell lines disclosed herein.

One aspect of the invention provides a vector for transfecting NK-92cells, wherein the vector includes a nucleic acid sequence encoding aprotein that is either a cytokine which promotes the growth of the NK-92cells, a cellular component responsive to an agent, a cancer cellreceptor molecule, or any combination of these proteins. Whentransfected with the vector, the NK-92 cells constitutively express theprotein. In an important embodiment, the protein is the cytokineinterleukin 2. In especially important embodiments of this aspect of theinvention, the vectors are MFG-hIL-2 and pCEP4-LTRhIL-2. In additionalsignificant embodiments, the protein is a cellular component responsiveto an agent, such that when the vector transfects NK-92 cells and theagent is taken up by the cells, the cells are inactivated. In still moresignificant embodiments the agent is either acyclovir or gancyclovir. Afurther embodiment of the invention provides a cell populationcontaining NK-92 cells that have been modified by a physical treatmentor by transfection with a vector.

In significant embodiments of this population, the physical treatmentrenders them non-proliferative yet does not significantly diminish thecytotoxicity of the cells, and in particularly significant embodiments,the treatment is irradiation. In additional important embodiments thecells have been transfected by a vector that encodes a cytokinepromoting the growth of the cells. The cells secrete the cytokine bothupon being cultured under conditions that promote cytokine secretion orin vivo upon being introduced into a mammal. In particularly importantembodiments of this aspect of the invention, the cytokine is interleukin2. In still further important embodiments, the NK-92 cells are the cellsNK-92MI, modified by transfection with the vector MFG-hIL-2 encodinginterleukin-2, and the cells NK-92CI modified by transfection with thevector pCEP4-LTRhIL-2 encoding interleukin-2. The NK-92MI cell line wasdeposited into the general depository of the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, onSep. 3, 1998, was assigned ATCC Deposit No. CRL-2408, was moved to thepatent depository on Apr. 4, 2005, and was assigned Deposit No.PTA-6671. The NK-92CI cell line was deposited into the generaldepository of ATCC on Sep. 3, 1998, was assigned ATCC Deposit No.CRL-2409, was moved to the patent depository on Apr. 4, 2005, and wasassigned ATCC Deposit No. PTA-6672. In additional important embodiments,the NK-92 cells are transfected by a vector including a sequence thatencodes a cellular component responsive to an agent such that, when theNK-92 cell so transfected takes up the agent, the cell is inactivated.In particularly important embodiments thereof, the agent is acyclovir organcyclovir. In yet additional embodiments, the cell population istransfected with a vector encoding a cancer cell receptor molecule.

The present invention also provides a method of purging cells related toa pathology from a biological sample including the steps of (i)obtaining a biological sample from a mammal that is suspected ofcontaining cells related to the pathology, and (ii) contacting thesample with a medium comprising NK-92 or modified NK-92 natural killercells, wherein the modified NK-92 cells have been modified by a physicaltreatment or by transfection with a vector. In significant embodimentsof this method, the pathology is a cancer, or is an infection by apathogenic virus such as human immunodeficiency virus (HIV),Epstein-Barr virus (EBV), cytomegalovirus (CMV), or herpes virus. Inadditional important embodiments, the modified NK-92 cells haveundergone a physical treatment that renders them non-proliferative, yetwhich does not significantly diminish their cytotoxicity, or have beentransfected with a vector, or they have been treated by any combinationof these modifications. In significant embodiments of this method, thevector encodes a cytokine that promotes the growth of the cells, aprotein that is responsive to an agent, a cancer cell receptor molecule,or a combination of these coding sequences. In a further embodiment, themedium also includes a cytokine that promotes the growth of the cells.The sample, once purged of cancer cells, may be further treated,including, for example, being returned to the mammal from which it wasobtained. In important embodiments of the method, the biological sampleis blood or bone marrow, the mammal is a human, and/or the naturalkiller cell is immobilized on a support.

The invention additionally provides a method of treating a pathology exvivo in a mammal including the steps of (i) obtaining a biologicalsample suspected of containing cells related to the pathology from themammal; (ii) contacting the biological sample with a medium includingnatural killer cells, either NK-92 cells or modified NK-92 cells thathave been modified by a physical treatment or by transfection with avector, thereby selectively destroying the cells related to thepathology in the sample and producing a purged sample, and (iii)returning the purged sample to the mammal. The pathology may be acancer, such as a leukemia, a lymphoma, or a multiple myeloma.Alternatively, the pathology may be infection by a pathogenic virus suchas HIV, EBV, CMV, or herpes. In this method the natural killer cells maybe NK-92 itself or modified NK-92 cells. Examples of such modified NK-92cells include those that have been modified by a physical treatment thatrenders them non-proliferative yet does not significantly diminish theircytotoxicity, and modification by transfection with a vector. The vectorencodes a cytokine that promotes the growth of the cells, or a proteinthat is responsive to an agent, or a cancer cell receptor molecule, orthe vector may include any combination of these modifications. Inimportant embodiments of this method, the biological sample is blood orbone marrow, the mammal is a human, and/or the natural killer cell isimmobilized on a support. In additional significant embodiments, themedium further includes a cytokine that promotes the growth of thecells, and/or the cancer is a leukemia, a lymphoma or a multiplemyeloma.

The present invention further provides a method of treating a pathologyin vivo in a mammal including the step of administering to the mammal amedium comprising natural killer cells, either NK-92 cells or NK-92cells that have been modified by a physical treatment that renders themnon-proliferative yet does not significantly diminish theircytotoxicity, by treatment that inhibits expression of HLA antigens onthe NK-92 cell surface, or by transfection with a vector. The vectorencodes a cytokine that promotes the growth of the cells, or a proteinthat is responsive to an agent, or a cancer cell receptor molecule, orthey have been treated by any combination of these modifications. Inimportant embodiments, the pathology is a cancer, such as a leukemia, alymphoma, or a multiple myeloma. Alternatively, in important embodimentsthe pathology is infection by a pathogenic virus such as HIV, EBV, CMV,or herpes. Advantageous embodiments of this method include administeringthe cells intravenously to a human and administering a cytokine thatpromotes the growth of the cells to the mammal in conjunction withadministering the medium comprising the natural killer cell. The presentmethods are especially adapted for the treatment of leukemia, lymphomaor multiple myeloma.

In yet an additional embodiment of the in vivo method of treatingcancer, the NK-92 is modified by transfection with a vector comprisingan element responsive to an agent such that when the agent is taken upby the cell, the cell is inactivated. According to this method, anamount of the agent effective to inactivate the cell can be administeredto a mammal after a time sufficient for the natural killer cell to treatthe cancer has elapsed or at a time desirable to effectively end thetreatment. A significant aspect of this embodiment is one in which theagent is acyclovir or gancyclovir. Such transfected cells can, ineffect, be “turned off” as desired by administering the agent.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Cytotoxic activity of NK-92 against different leukemic targetcell lines tested in a 4 hour ⁵¹Cr release assay. The results representthe mean±the standard deviation (SD) for three replicate experiments.

FIG. 2. Cytotoxicity of NK-92 after IL-2 deprivation. NK-92 cells werecultured in enriched alpha medium (Myelocult™, StemCell Technologies,Vancouver, BC) without IL-2. Cytotoxicity was measured daily with the⁵¹Cr-release assay against K562-neo′ or Daudi target cells. The Figureshows results from one representative experiment at the E:T ratio of10:1.

FIG. 3. Effect of various doses of γ radiation on the cytolyticpotential of NK-92 cells. NK-92 cells were irradiated with a ¹³⁷Cssource using doses ranging from 200 to 1000 cGy. To allow for recovery,cells were left in medium containing IL-2 for 24 hours beforecytotoxicity was measured in a 4 hour ⁵¹Cr release assay against thetarget cell line K562.

FIG. 4. Survival curves of NK-92 cells after γ-irradiation. NK-92 cellswere irradiated with a γ ray source at doses of 300, 500, 1000, and 3000cGy. Viability of NK-92 cells was determined by trypan blue staining.The maximal achievable concentration of the non-irradiated NK-92 cellsin culture was about 1.5×10⁶/mL. The cells had to be fed to preventovergrowth.

FIG. 5. Effect of γ-irradiation on the in vitro colony formation ofNK-92 cells. NK-92 cells were cultured in agar-based medium supplementedwith recombinant human IL-2 (rhIL-2).

FIG. 6. Effect of various radiation doses on the cytolytic potential ofNK-92 cells. NK-92 cells were γ-irradiated at doses of 300, 500, 1000,and 3000 cGy. ⁵¹Cr-labeled leukemic target cells K562 (Panel A) and HL60(Panel B) as well as two patient-derived leukemic samples TA27 (Panel C)and BA25 (Panel D) were tested for susceptibility to cytolysis byirradiated and non-irradiated (NR) NK-92 cells. The results of 4 hrchromium release assays are expressed as 30% lytic units/10⁸ effectorcells.

FIG. 7. Selective killing of patient-derived leukemic cells by NK-92cells. ⁵¹Cr-labeled leukemic target cells derived from 40 patients [9acute myeloid leukemia (AML) cases, 11 chronic myeloid leukemia (CML)cases, 14 B-lineage-acute lymphoblastic leukemia (ALL) cases and 6 T-ALLcases] and T cell depleted normal bone marrow cells from 14 normaldonors were tested for susceptibility to cytolysis by NK-92 cells atfour different E:T ratios. The results of a 4 hr chromium release assayare expressed as 30% lytic units/10⁸ effector cells.

FIG. 8. In vitro (Panels A and B) and in vivo (Panels C and D)antileukemic efficacy of NK-92 cells against K562 and HL60 leukemias ascompared to human LAK cells and other effectors. ⁵¹Cr labeled K562(Panel A) and HL60 (Panel B) cells were tested for susceptibility tocytolysis by NK-92 cells in comparison with various known effector cells[LAK, NK (CD3⁻ CD56⁺), and T cells (CD3⁺CD56⁻)] at indicated E:T ratiosin a 4 hr CRA assay. Results are means±SD of three separate tests forNK-92 cells, and two tests of different donor-derived effectors for LAK,CD56⁺ and CD3⁺ cells. SCID mice were inoculated subcutaneously with K562cells (Panel C) or HL60 cells (Panel D) (5×10⁶ cells per mouse) alone orin combination with NK-92, LAK, or NK cells at a 4:1 E:T ratio. As ameasure of the tumor sizes, their surface areas were measured once aweek post inoculation (n=5).

FIG. 9. Antileukemic effect of NK-92 cells, allogeneic cytotoxic Tlymphocyte (CTL) cells and other effector cells against apatient-derived acute T lymphoblastic leukemia (T-ALL) determined invitro and in vivo. Panel A: In vitro specific killing of T-ALL (TA27)target cells by NK-92, CTL, and other effector cells, was determined bya 4 hr ⁵¹Cr-release assay using the indicated E:T ratios. Results aremeans±SD of two or three separate tests. Panel B: SCID mice wereinoculated subcutaneously with TA27 cells (5×10⁶ each mouse) alone orco-inoculated with NK-92, CTL or other effector cells at a 4:1 E:Tratio. Recombinant human IL-2 (rhIL-2) was administered to the miceintraperitoneally for two weeks at the dose of 5×10⁴ U every other day.Leukemic tumor areas were measured once a week post inoculation (n=5).

FIG. 10. Survival of SCID mice bearing T-ALL (TA27) leukemiaco-inoculated with NK-92 cells as compared with co-inoculation withallogenic CTL or irradiated TALL-104 cells.

FIG. 11. Survival of SCID mice bearing T-ALL (TA27) after treatment withNK-92 cells. Mice received 5×10⁶TA27 cells intraperitoneally (I.P.).NK-92 cells (2×10⁷) were injected I.P. once, or 5 times (on days 1, 3,5, 7 and 9), with or without the addition of rhIL-2 every other day fortwo weeks.

FIG. 12. Survival of SCID mice bearing pre-B-ALL (BA31) after treatmentwith NK-92 cells. Mice received 5×10⁶ BA31 cells I.P. NK-92 (2×10⁷)cells were injected I.P. for a total of 5 doses, on days 1, 3, 5, 7 and9.

Mice in the indicated groups received rhIL-2 every other day for twoweeks.

FIG. 13. Survival of SCID mice bearing human AML (MA26) after treatmentwith NK-92 cells. Mice received 5×10⁶ MA26 leukemia cells I.P. NK-92(2×10⁷) cells were injected I.P. on days 1, 3, 5, 7 and 9 for a total offive doses. Mice in the indicated groups received rhIL-2 every other dayfor two weeks.

FIG. 14. Diagrammatic map of plasmid MFG-hIL-2.

FIG. 15. Diagrammatic map of plasmid pCEP4-LTR.hIL-2.

FIG. 16. Cytotoxicity of NK-92, NK-92MI and NK-92CI against K562 andRaji target cells. The cytotoxic activities of the IL-2 transfectantswere compared to that of the parental cell line. NK cells were mixedwith ⁵¹Cr-labeled K562 (Panel A) or Raji (Panel B) cells ateffector:target ratios of 1:1, 5:1, 10:1 and 20:1 for a 4 hour chromiumrelease assay. The cytotoxicities of NK-92 (•), NK-92MI (A) and NK-92CI(▪) are shown.

FIG. 17. Effect of NK-92 MI and NK-92CI on hematopoietic progenitors. Toassay the effect of the NK-92 cells on normal hematopoietic progenitors,a clonogenic assay was performed. Normal PBMCs were incubated withirradiated NK-92MI or NK-92CI at various NK:PBMC ratios ranging from 1:1to 1:1000 for 48 hours. The cells were plated in methylcellulose atconcentrations to give 10-100 colonies per dish after 14 days.Clonogenic output of PBMCs incubated with NK-92MI (white bars) andNK-92CI (gray bars) is expressed as either total number of colonies orsubclassified on the basis of colony type (BFU-E, CFU-GM and CFU-GEMM).

FIG. 18. Effect of irradiation on NK-92, NK-92MI and NK-92CIproliferation and viability. To assess the effect of irradiation on theparental and transfected NK-92 cells, cells were exposed to 0, 500,1,000, 1,500, and 2,000 cGy doses of radiation and assayed forproliferation by a standard ³H-thymidine incorporation assay. Panel A:Proliferation of NK-92 (•), NK-92MI (▴) and NK-92CI (▪) is expressed asa percentage of control (unirradiated cells). Panel B: Cells wereexposed to 0, 250, 500, 1,000, and 2,000 cGy of irradiation and assessedby trypan blue exclusion for viability after 24 (black bars), 48 (graybars) and 72 hours (white bars).

FIG. 19. Effect of irradiation on NK-92, NK-92MI and NK-92CIcytotoxicity. To assess the effect of irradiation on cytotoxicity of theNK cells, NK-92, NK-92MI and NK-92CI were irradiated at 0, 1,000, and2,000 cGy and tested after three days for cytotoxicity against K562(Panel A) and Raji (Panel B) cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of treating a biological sampleor a mammal suspected of having a pathology such as a cancer or aninfection by a virus. Certain natural killer cells which are cytolyticfor the cells affected by the pathology are employed. The treatmentresults in significant diminution of the number, or, in some cases, theelimination, of malignant or cancerous cells, or virus-infected cells,in the sample or mammal. The natural killer cells of this invention aredesignated NK-92 cells and include certain treated or transfectedmodifications of NK-92 cells. These cells are highly effective inpurging cancer cells ex vivo and in destroying cancer cells in vivo.

As used in the present invention, “cytotoxic T lymphocytes” (CTL) relateto immune cells which kill antigen-specific target cells. CTL are MHCclass I-restricted. As used in the present invention, lymphokineactivated killer (LAK) cells relate to cells of the immune system thathave antitumor killing activity. They are obtained from a population ofcells, such as peripheral blood mononuclear cells, upon activation bytreatment with lymphokines. LAK cells have essentially no effect onnormal cells.

As used to describe the present invention, “natural killer (NK) cells”are cells of the immune system that kill target cells in the absence ofa specific antigenic stimulus, and without restriction according to MHCclass. Target cells may be tumor cells or cells harboring viruses. NKcells are characterized by the presence of CD56 and the absence of CD3surface markers. The present invention is based on an immortal NK cellline, NK-92, originally obtained from a patient having non-Hodgkin'slymphoma. As used to describe the present invention, a modified NK-92cell is an NK-92 cell which has been further treated to endow it withproperties not found in the parent from which it is derived. Suchtreatments include, for example, physical treatments, chemical and/orbiological treatments, and the like. The treatments confer propertiesupon the modified NK-92 cells that render them more advantageous for thepurposes of the invention.

As used to describe the present invention, the terms “cytotoxic” and“cytolytic”, when used to describe the activity of effector cells suchas NK cells, are intended to be synonymous. In general, cytotoxicactivity relates to killing of target cells by any of a variety ofbiological, biochemical, or biophysical mechanisms. Cytolysis refersmore specifically to activity in which the effector lyses the plasmamembrane of the target cell, thereby destroying its physical integrity.This results in the killing of the target cell. Without wishing to bebound by theory, it is believed that the cytotoxic effect of NK cells isdue to cytolysis.

As used to describe the present invention, “target cells” are the cellsthat are killed by the cytotoxic activity of the NK cells of theinvention. These include in particular cells that are malignant orotherwise derived from a cancer, and cells that are infected bypathogenic viruses such as HIV, EBV, CMV, or herpes.

As used to describe the present invention, “purging” relates to killingof target cells by effector cells such as NK cells ex vivo. The targetcells may be included in a biological sample obtained from a mammalbelieved to be suffering from a pathology related to the presence of thetarget cell in the sample. The pathology may be a cancer or malignancydue to tumor cells in the sample, and may be treated by purging thesample of the tumor cells and returning the sample to the body of themammal.

As used to describe the present invention, “inactivation” of the NK-92cells renders them incapable of growth and/or their normal function, inparticular, their cytotoxic activity. Inactivation may also relate tothe death of the NK-92 cells. It is envisioned that the NK-92 cells maybe inactivated after they have effectively purged an ex vivo sample ofcells related to a pathology in a therapeutic application, or after theyhave resided within the body of a mammal a sufficient period of time toeffectively kill many or all target cells residing within the body.Inactivation may be induced, by way of nonlimiting example, byadministering an inactivating agent to which the NK-92 cells aresensitive.

As used herein, a “vector” relates to a nucleic acid which functions toincorporate a particular nucleic acid segment, such as a sequenceencoding a particular gene, into a cell. In most cases, the cell doesnot naturally contain the gene, so that the particular gene beingincorporated is a heterologous gene. A vector may include additionalfunctional elements that direct and/or regulate transcription of theinserted gene or fragment. The regulatory sequence is operablypositioned with respect to the protein-encoding sequence such that, whenthe vector is introduced into a suitable host cell and the regulatorysequence exerts its effect, the protein is expressed. Regulatorysequences may include, by way of non-limiting example, a promoter,regions upstream or downstream of the promoter such as enhancers thatmay regulate the transcriptional activity of the promoter, and an originof replication. A vector may additionally include appropriaterestriction sites, antibiotic resistance or other markers for selectionof vector containing cells, RNA splice junctions, a transcriptiontermination region, and so forth.

As used to describe the present invention, “cancer”, “tumor”, and“malignancy” all relate equivalently to a hyperplasia of a tissue ororgan. If the tissue is a part of the lymphatic or immune system,malignant cells may include non-solid tumors of circulating cells.Malignancies of other tissues or organs may produce solid tumors. Ingeneral, the methods of the present invention may be used in thetreatment of lymphatic cells, circulating immune cells, and solidtumors.

As used to describe the present invention, a “pathogenic virus” is avirus causing disease in a host. The pathogenic virus infects cells ofthe host animal and the consequence of such infection is a deteriorationin the health of the host. Pathogenic viruses envisioned by the presentinvention include, but are not limited to, HIV, EBV, CMV, and herpes.

Natural Killer Cell NK-92.

The NK-92 cell line has been described by Gong et al. (1994). It isfound to exhibit the CD56^(bright), CD2, CD7, CD11a, CD28, CD45, andCD54 surface markers. It furthermore does not display the CD1, CD3, CD4,CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growthof NK-92 cells in culture is dependent upon the presence of recombinantinterleukin 2 (rIL-2), with a dose as low as 10 IU/mL being sufficientto maintain proliferation. IL-7 and IL-12 do not support long-termgrowth, nor do other cytokines tested, including IL-1α, IL-6, tumornecrosis factor α, interferon α, and interferon γ. NK-92 is highlyeffective in killing certain tumor cells, such as K562 (erythroleukemia)and Daudi (Burkitt lymphoma) cells, for it has high cytotoxicity even ata low effector:target (E:T) ratio of 1:1 (Gong et al. (1994)). Inaddition, NK-92 cells have high cytotoxic activity against 8E5 cells,which are infected with HIV and produce HIV virions. NK-92 cells weredeposited into the general despository of the American Type CultureCollection (ATCC), 10801 University Blvd., Masassas, Va. 20110-2209, onSep. 3, 1998, was assigned ATCC Deposit No. CRL-2407, was moved to thepatent depository on Apr. 4, 2005, and was assigned ATCC Deposit No.PTA-6670.

NK-92 cells are readily maintained in culture medium, such as enrichedalpha minimum essential medium (MEM; Sigma Chemical Co., St. Louis, Mo.)supplemented with fetal calf serum (for example, at 12.5%; SigmaChemical Co., St. Louis, Mo.), and horse serum (for example, at 12.5%;Sigma Chemical Co., St. Louis, Mo.). Initially, 10⁶ M hydrocortisone isrequired, but in subsequent passages it is found that hydrocortisone maybe omitted. In addition, IL-2, such as recombinant human IL-2 (500 U/mL;Chiron, Emeryville, Calif.), is required for long-term growth. Whensuspension cultures are maintained in this fashion with semiweeklychanges of medium, the cells exhibit a doubling time of about 24 h.

NK-92 cells in vitro demonstrate lytic activity against a broad range ofmalignant target cells. These include cell lines derived fromcirculating target cells such as acute and chronic lymphoblastic andmyelogenous leukemia, lymphoma, myeloma, melanoma, as well as cells fromsolid tumors such as prostate cancer, neuroblastoma, and breast cancercell lines. This effect is observed even at very low effector:targetratios. This lysis is superior to cytotoxicity obtained from normalperipheral blood mononuclear cells stimulated for four days with IL-2.

Vector for Transfecting Mammalian Cells to Produce Cytokine.

The present invention provides NK-92 cells which have been modified bytransfection with a vector that directs the secretion of a cytokine,such as IL-2. In order that NK-92 cells maintain long-term growth andcytolytic function, they generally must be supplied with IL-2. A vectorencoding the gene for human IL-2, and which also contains a controlelement directing the synthesis of the IL-2 gene product is therefore ofgreat utility in the invention. NK-92 cells bearing such a vectorsecrete the IL-2 needed for cytolytic activity in a therapeutic setting;thus, IL-2 from an exogenous source is not required. The control elementis one which directs the synthesis of IL-2 as a constitutive product,i.e., one that is not dependent upon induction. Methods for constructingand employing vectors are described in general terms in “CurrentProtocols in Molecular Biology”, Ausubel et al., John Wiley and Sons,New York (1987, updated quarterly), and “Molecular Cloning: A LaboratoryManual 2nd Ed.”, Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989), which are incorporatedherein by reference.

Modified NK-92 Transfected to Produce Cytokine, and Method ofTransfecting.

Modified NK-92 cells that secrete a cytokine may be prepared byinserting a vector that directs the synthesis and secretion of thecytokine into the cells. In important aspects of the invention, thecytokine is IL-2. Methods of introducing a vector into a mammalian cellare well known to workers of ordinary skill in molecular biology andcellular immunology, and are described in Ausubel et al. (1987, updatedquarterly) and Sambrook et al. (1989). The vectors encoding the cytokineencompass as well control elements that lead to constitutive synthesisof the cytokine when incorporated into the NK-92 cells.

When cultured under appropriate conditions that promote cytokinesecretion the transfected NK-92 cells secrete IL-2 or other cytokine.Since the vector directs constitutive synthesis of the cytokine,nutrient cultures in which the NK-92 cells are known to grow and toexhibit their normal cytolytic function are sufficient for thetransfected cells to secrete the cytokine. For the same reason, thetransfected cells secrete the cytokine in vivo when they are introducedwithin the body of a mammal.

NK-92 cells transfected with a vector that directs secretion of acytokine such as IL-2 are useful in the ex vivo treatment of abiological sample drawn from a mammal which is suspected of containingmalignant cells. By treating the malignant cells with these modifiedNK-92 cells, the need for applying exogenous IL-2 or other cytokine isobviated. These modified NK-92 cells are useful, for the same reasons,in the in vivo treatment of a mammal suffering from a malignancy. Themodified NK-92 cells exert their cytolytic effect against the malignantcells when introduced into the body of the mammal. Examples of suchcells in the present invention are designated NK-92MI and NK-92CI.

NK-92 Cells that are Cytolytic but not Capable of Proliferation.

An additional modified NK-92 cell of the invention is one that has beentreated in such a way that it is no longer able to proliferate, yetwhose cytotoxic activity is preserved. One way of achieving this stateis by γ irradiation. Additional forms of radiation, including, forexample, ultraviolet radiation, may be employed. Suitable sources to usefor this purpose include, for example, a ¹³⁷Cs source (Cis-US, Bedford,Mass.; Gammacell 40, Atomic Energy of Canada Ltd., Canada).Additionally, proliferative activity may be abrogated by treatment withchemical agents which inhibit DNA synthesis. An example of such an agentis mitomycin C.

Vector for Transfecting NK-92 with an Element Responsive to anInactivating Agent.

The NK-92 cells may also be modified by transfection with a vector suchthat, when the cell takes up a specific agent, the cell is inactivated.The vector includes a sequence that encodes a cellular componentresponsive to the agent, such that when the vector transfects a cell andthe agent is taken up by the cell, the cell is inactivated. In preferredembodiments, the agent is acyclovir or gancyclovir. The vector alsocontains a control element directing the synthesis of the cellularcomponent as a constitutive product.

The NK-92 cell transfected with the vector described in the precedingparagraph maintains its characteristic growth and cytolytic activity inthe absence of the agent. At a point in time, for example, when an exvivo sample has been purged of malignant cells by the action of theNK-92 cells, or when the NK-92 cells administered in vivo haveeffectively exerted their cytolytic activity within a mammalian body, orwhen it desired that the treatment be stopped for any reason, the agentmay be administered. The agent interacts with the cellular componentsensitive to the agent encoded in the vector. The interaction of theagent with the cellular component induces the inactivation of the NK-92cells. Inactivation may range from loss of characteristic cytolyticfunction to death of the cells.

This property of the modified NK-92 cells is significant because theparent NK-92 cells are derived from a tumor cell line that may continuepropagating in a sample reintroduced into a mammal after ex vivotherapy, or in vivo when so administered. It is therefore important toablate the cells after they have carried out their therapeutic function.Rendering the cells sensitive to an agent such as acyclovir organcyclovir is an advantageous way of achieving this objective.

Vector for Transfecting NK-92 with an Altered HLA Cell Surface Molecule.

The HLA cell surface protein, involved in presenting antigens to othercells of the immune system, includes a non-immunospecific subunit, theprotein β₂-microglobulin. If this protein is altered or mutated, the HLAprotein loses its affinity for the T-cell receptor to which itordinarily binds. The β₂-microglobulin gene in NK-92 cells of theinvention may be mutated by site specific mutagenesis in order totransform its properties in this way. The result is an NK-92 cell whichno longer has a high affinity for T-cell receptors. As a result, theNK-92 cell modified in this way remains within the host organism for alonger period of time, rather than being eliminated by the action of thehost's cellular immune response.

Vector for Transfecting NK-92 with a Gene Encoding a Cancer CellReceptor Molecule.

The NK-92 cells may also be modified by transfection with a vector suchthat the cells constitutively express a receptor for a cancer cell.Cancer cells express cell surface molecules that are idiosyncratic forthe origin of the cancer, and frequently are also idiosyncratic for theindividual host. The CTL population in such diseased patients may havebeen activated by exposure to the cells of the growing cancer. Suchactivated CTL express cell surface proteins that are specific for, ortarget, the cells of the cancer. These CTL may be isolated, the gene forthe targeting receptor identified, isolated, and transfected into theNK-92 cells of the invention. This confers on the NK-92 cells thecapability of likewise specifically targeting the cancer cells presentin the individual host. This has the effect of enhancing the specificityof the cytotoxic activity of the NK-92 cells toward the cancer cells ofthat individual. The corresponding process would be carried out for eachhost suffering from cancer, taking advantage of the idiosyncraticspecificity of the CTL targeting moiety in each case.

Methods of Treating.

The natural killer cells of the invention are employed in methods oftreating biological samples in order to purge them of cells from acancer, a malignancy, or a tumor, or cells infected by a pathogenicvirus. The NK cells include by way of nonlimiting example, NK-92, andmodified NK-92 cells, such as NK-92MI and NK-92CI, as well as othermodified NK-92 cells envisioned within the scope of this invention. TheNK-92MI and NK-92CI cells are modified by transfection with vectors thatresult in the secretion of IL-2. In addition, any of the NK-92, NK-92MI,and NK-92CI cells may be treated such that they maintain the cytolyticactivity of the untreated cells but cannot proliferate. The NK cells sotreated may also be equivalent cell lines which have the properties suchas cytotoxicity and NK-specific cell surface markers described herein.Malignancies of the immune system, the lymphatic system, and thehematopoietic system may be treated by the methods of the invention. Inaddition, formed tumors and solid tumors may also be treated. Infectionsby pathogenic viruses, such as HIV, EBV, CMV, and herpes may also betreated.

Treating a Biological Sample.

In vitro biological samples may be treated experimentally ortherapeutically in order to eliminate malignant cells, or virus-infectedcells, in an effective manner. The sample may be drawn from a mammal andmaintained in vitro in an appropriate culture medium. Such media arewell known to workers of skill in cell biology, cellular immunology, andoncology. Media and cell culture techniques are presented in generalterms in, for example, Freshney, R. I., “Culture of Animal Cells, 3rdEd.”, Wiley-Liss, New York (1994), and in Martin, B. M., “Tissue CultureTechniques, An Introduction”, Birkhauser, Boston, Mass. (1994), whichare incorporated herein by reference. The biological sample isestablished in culture in vitro, and contacted with a medium thatincludes the natural killer cells of the present invention. Thecytolytic activity of the NK cells effectively eliminates the malignantcells or the virus-infected cells from the sample. The prevalence anddepletion of the target cells may be traced by any of a number ofmethods well known to those of skill in the fields of cell biology andcellular immunology. These include indirect immunofluorescencemicroscopy to assay for intact tumor cells or virus-bearing cells,fluorescent-activated cell sorting, chromium release assays, and thelike.

Treating a Cancer or Virus Infection Ex Vivo: Purging.

The present invention additionally encompasses the ex vivo treatment ofa biological sample suspected of containing cancer cells orvirus-infected cells by contacting the sample with the NK cells of theinvention. The biological sample is drawn from the body of a mammal,such as a human, and may be blood, bone marrow cells, or similar tissuesor cells from an organ afflicted with a cancer. Methods for obtainingsuch samples are well known to workers in the fields of cellularimmunology, oncology, and surgery. They include sampling blood in wellknown ways, or obtaining biopsies from the bone marrow or other tissueor organ. The cancer cells or virus-infected cells contained in thesample are effectively eliminated due to the cytotoxic activity of theNK-92 cells. The sample may then be returned to the body of the mammalfrom which it was obtained.

The NK-92 cells used to treat the sample, may be freely suspended in themedium. It is generally preferred that the purged sample, prior to beingreturned to the body of the mammal from which it was obtained, be rid ofNK-92 cells that may continue growing, since they arose originally froma proliferating lymphoma. The invention envisions several ways ofaccomplishing this objective. In one embodiment, the NK cells, prior touse, are irradiated with γ rays or with ultraviolet light to the extentthat they maintain their cytolytic activity but are not capable ofgrowth. In an additional embodiment, the NK cells are permanentlyimmobilized on a macroscopic solid support. The support with the NKcells attached may then be physically separated from the cells of thebiological sample, for example by centrifugation, or filtration with acolumn which permits the unbound cells of the sample to pass through, orlike technique. Suitable solid supports include particles ofpolyacrylamide, agarose, cellulose, Sepharose™ (Pharmacia, Piscataway,N.J.), celite, and the like, and may be supplied with groups such as analdehyde, carbonyldiimidazole, broamoacetyl, epichlorhydrin, and thelike, which are activated for reaction with cell surface groups. Theactivated groups on the support react with groups such as amino orcarboxyl groups, for example, on the cell surface, thereby immobilizingthe cells on the support.

In yet a further embodiment, the NK cells may be modified with a vectordirecting the synthesis of a cellular component sensitive to an agent,such that when the agent is administered to the ex vivo sample, theNK-92 cells are inactivated. Examples of such agents include acycloviror gancyclovir, by way of nonlimiting example. Functionally equivalentvectors, directing the synthesis of alternative cellular componentssensitive to different agents, are also envisioned within the scope ofthis embodiment.

The NK cells to be used in the methods of the invention may require acytokine such as IL-2 to maintain their functional effectiveness ascytolytic cells. The cytokine may simply be added to the ex vivopreparation. Alternatively, if desired, a modified NK-92 cell bearing avector directing the constitutive synthesis of the cytokine may beemployed. In this way the necessity of furnishing exogenous cytokine isavoided.

Treating a Cancer or Virus Infection In Vivo: Administering NK-92.

A further method of the invention is directed toward treatment of acancer or a virus infection in vivo in a mammal using NK-92 cells. Thecells are administered in a variety of ways. By way of nonlimitingexample, the cells may be delivered intravenously, or into a body cavityadjacent to the location of a solid tumor, such as the intraperitonealcavity, or injected directly within or adjacent to a solid tumor.Intravenous administration, for example, is advantageous in thetreatment of leukemias, lymphomas, and comparable malignancies of thelymphatic system, as well as in the treatment of viral infections.

As has been described in detail in the preceding section, it isdesirable to employ methods that eliminate or ablate the NK-92 cellsafter they have effectively lysed (or otherwise destroyed) the targetcells. Certain methods described above may be employed for this purpose,namely, use of irradiated NK-92 cells, and use of NK-92 cells harboringa vector directing the synthesis of a cellular component sensitive to anagent, such that when the agent is administered, the NK-92 cells areinactivated, and equivalent methods. When the cells produce such acomponent sensitive to the specific agent, administration of the agentto the mammal is effective to inactivate the NK-92 cells within themammal.

The NK-92 cells may be administered in conjunction with a cytokine suchas IL-2 in order to maintain the functional effectiveness of the cellsas cytotoxic effectors. As used to describe the invention, the term “inconjunction” indicates that the cytokine may be administered shortlyprior to administration of the NK-92 cells, or it may be givensimultaneously with the cells, or shortly after the cells have beenadministered. The cytokine may also be given at two such times, or atall three times with respect to the time of administering the NK-92cells. Alternatively, NK-92 cells harboring a vector directing theconstitutive synthesis of the cytokine may be employed in the in vivomethod of treating a cancer. This effectively eliminates the need tofurnish exogenous cytokine.

The following examples are included to illustrate the invention and notto limit the invention. All publications or references cited in thepresent specification are hereby incorporated by reference.

EXAMPLES Example 1. NK-92 Cells

NK-92 cells (Gong et al. (1994)) were derived from cells obtained from apatient suffering from non-Hodgkin's lymphoma. PBMC from the patientwere cultured in enriched alpha MEM supplemented with fetal calf serum(12.5%) and horse serum (12.5%) plus 10-⁶M hydrocortisone and 1000 U/mLof recombinant human IL-2 (rhIL-2). Cells were cultured at 37° C. inhumidified air containing 5% CO₂. Subcultures were made after 4 weeks,and propagated indefinitely with twice-weekly changes in medium. Inthese later stages the hydrocortisone could be omitted without anyeffect on cell growth. This culture has been designated NK-92 and hasbeen deposited with the American Type Culture Collection (ATCC;Rockville, Md.) under designation CLR-2407.

The cells have the morphology of large granular lymphocytes. The cellsbear the CD56^(bright), CD2, CD7, CD11a, CD28, CD45, and CD54 surfacemarkers. In contrast, they do not display the CD1, CD3, CD4, CD5, CD8,CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growth of NK-92cells in culture is dependent upon the presence of recombinantinterleukin 2 (IL-2), with a dose as low as 10 IU/mL being sufficient tomaintain proliferation. IL-7 and IL-12 do not support long-term growth,nor do other cytokines tested, IL-1α, IL-6, tumor necrosis factor α,interferon α, and interferon γ.

Example 2. Cytotoxic Activity of NK-92 Against Different Leukemic CellLines

The cytotoxic activity of NK-92 against K562, Daudi, TF-1, AML-193, andSR-91 cells was determined (Gong et al. (1994)). K562 (erythroleukemia)and Daudi (Burkitt) lymphoma cell lines were obtained from ATCC. Theywere maintained in continuous suspension culture in RPMI 1640 mediumsupplemented with 10% fetal calf serum (FCS). TF-1 is a myelomonocyticcell line (Kitamura et al., J. Cell Physiol. 140:323-334 (1989)) thatrequires the presence of medium containing 2 ng/mL of human GM-CSF.AML-193 is a myeloid cell line that is maintained in the presence of 10%5637-conditioned medium (Lange et al., Blood 70:192-199 (1987)). BothTF-1 and AML-193 cells were obtained from Dr. D. Hogge, Terry FoxLaboratory, University of British Columbia, Vancouver, BC. SR-91 is acell line with features of early progenitor cells established by Gong etal. (1994) from a patient with acute lymphoblastic leukemia (ALL)(Klingemann et al., Leuk. Lymphoma, 12, 463-470 (1994). It is resistantto both NK and activated-NK (A-NK) cell cytotoxicity. SR-91 is alsomaintained in RPMI 1640/10% FCS. This cell line can be renderedsensitive to killing by NK-92 by treatment with cytokine. Naki et al.,“Induction of sensitivity to the NK-mediated cytotoxicity by TNF-αtreatment: Possible role of ICAH-3 and CD44,” Leukemia, in press.

The cytotoxic activity of NK-92 (effector) against these target cellswas assessed by means of a ⁵¹Cr release assay (Gong et al. (1994)) usingthe procedure described by Klingemann et al. (Cancer Immunol.Immunother. 33:395-397 (1991)). The percentage of specific cytotoxicityin triplicate specimens was calculated as:

${\%\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{release}} = {\frac{\left( {{{average}\mspace{14mu}{experimental}\mspace{14mu}{cpm}} - {{average}\mspace{14mu}{spontaneous}\mspace{14mu}{cpm}}} \right)}{\left( {{{average}\mspace{14mu}{maximum}\mspace{14mu}{cpm}} - {{average}\mspace{14mu}{spontaneous}\mspace{14mu}{cpm}}} \right)} \times 100}$FIG. 1 presents the results of this determination. It is seen that NK-92cells kill K562 and Daudi cells with high efficiency. Even at the lowE:T ratio of 1:1, 83% of K562 cells and 76% of Daudi cells were killedby NK-92 cells. Susceptibility to killing by NK-92 cells was lower forTF-1. cells (23% at E:T=1:1) and for AML-193 cells (6% at E:T=1:1).SR-91 cells appear to be resistant to the cytotoxic effect of the NK-92cells. Without wishing to be bound by theory, it is believed that SR-91cells lack adhesion molecules necessary to mediate initial binding withNK-92 cells.

Example 3. Cytotoxicity of NK-92 Against Leukemia, Lymphoma, and MyelomaTarget Cell Lines

K562 (Ph-chromosome positive [Ph*] erythroleukemia), HL60(promyelocytic), U937 (myelomonocytic), KG1 a (variant subline of theAML cell line KG1), DHL-10 (B-cell lymphoma), Daudi (Burkitt'slymphoma), Raji (B-cell lymphoma), Jurkat (T-cell lymphoma), 0266 (IgEmyeloma), NCI H929 (IgA myeloma), and RPMI 8226 (myeloma, light chainsecreting) cell lines were obtained from ATCC. The lymphoma-derived celllines Ly3 (B-lineage, diffuse large cell), Ly8 (immunoblastic), andLy13.2 (T-lineage, diffuse large cell) were provided by Dr. H. Messner,Toronto, Ontario. Their characteristics have been described (Chang etal., Leuk. Lymphoma 19:165 (1995)). All lines were maintained in RPM11640 medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1mM nonessential amino acids, 50 U/mL penicillin, 25 mM HEPES (StemCellTechnologies), and 5% heat-inactive FCS (RPMI/5% FCS) at 37° C. in ahumidified atmosphere of 5% CO₂ in air.

Cell lysis was determined by a 4-hour ⁵¹Cr release assay using variousE:T ratios. To allow for comparison, PBMCs from normal donors wereactivated with IL-2 (500 U/mL) for 4 days and ⁵¹Cr release measuredagainst the same target cells concurrently. The mean of two separateexperiments is presented.

Results of NK-92-mediated cytotoxicity (“Cr release assay) againstvarious leukemia, lymphoma, and myeloma target cell lines are summarizedin Table 1. For comparison, lysis of the same tumor target cells wasalso tested in the same experiment with PBMCs obtained from normaldonors. Those cells had been activated by IL-2 (500 U/mL) for 4 daysprior to testing. Results show that NK-92 cells very effectively lyseall target cells tested. High cytotoxicity is observed even at the lowE:T ratio of 1:1. The cytotoxicity achieved with these cells issignificantly higher than that observed with normal (allogeneic) PBMCsactivated under optimal conditions with IL-2 for all the target cellsexcept RPMI 8226 and 0266.

TABLE 1 Cytotoxic activity of NK-92 cells against various leukemia,lymphoma, and myeloma cell lines Target 50:1 20:1 10:1 5:1 1:1 HL-60NK-92 97 90 77 46 40 PBMCs + IL-2 31 26 17 2 0 K562 NK-92 68 68 64 59 50PBMCs + IL-2 63 73 67 51 19 KG1a NK-92 90 91 80 67 39 PBMCs + IL-2 15 1112 6 0 U937 NK-92 99 98 96 91 85 PBMCs + IL-2 57 43 23 13 2 DHL-10 NK-9295 95 92 94 80 PBMCs + IL-2 60 40 24 19 5 Daudi NK-92 94 87 71 48 39PBMCs + IL-2 65 57 29 16 6 Jurkat NK-92 100 100 98 93 80 PBMCs + IL-2 6750 36 27 4 Ly 3 NK-92 63 59 53 42 28 PBMCs + IL-2 47 35 18 6 095 Ly 8NK-92 95 104 102 88 42 PBMCs + IL-2 67 65 62 59 44 Ly 13.2 NK-92 104 105100 97 67 PBMCs + IL-2 61 63 52 4 13 Raji NK-92 81 75 74 70 54 PBMCs +IL-2 32 67 57 35 13 NCI H929 NK-92 94 89 89 86 51 PBMCs + IL-2 75 58 3924 5 RPMI NK-92 82 72 70 72 41 8224 PBMCs + IL-2 95 83 81 67 25 U266NK-92 84 77 85 81 53 PBMCs + IL-2 84 74 73 56 21

Example 4. Effect of Deprivation of IL-2 on Cytotoxic Activity of NK-92

To test how long NK-92 cells would maintain their cytolytic activitywithout IL-2 present in the culture medium, NK-92 cells were deprived ofIL-2 and ⁵¹Cr-release was measured in 24-hour intervals. Results,summarized in FIG. 2, suggest that the cells maintain full cytotoxicactivity for at least 48 hours. Thereafter, the activity dropsprecipitously to negligible levels. Thus, for short-term purging, IL-2does not have to be present in the cultures to achieve a suitableeffect.

Example 5. Co-Culture of K562-Neo′ Cells with PBMC's and NK-92

The transfection of the K562 cells with the neomycin-resistance (neo′)gene has been described (Wong et al., Bone Marrow Transplant 18:63(1966)). Briefly, 5×10⁷ K562 cells were suspended in 0.8 mL RPMI 1640/5%FCS and incubated on ice for 10 minutes with 30 μg of the pMCI-Neoplasmid (provided by Dr. K. Humphries, Terry Fox Laboratory, Vancouver,BC). The cells were then exposed to a single voltage pulse (125 μF/0.4kV) at room temperature, allowed to remain in buffer for 10 minutes,transferred into 25-cm² tissue culture flasks (Falcon, Lincoln Park,N.J.), and incubated at 37° C. in a humidified atmosphere of 5% CO₂ inair for 2 days. Transfected cells were selected in 0.8% Iscove'smethylcellulose medium (StemCell Technologies) supplemented with 30%FCS, 10⁻⁴ M 2-mercaptoethanol, and 2 mM glutamine, containing 0.8 mg/mLG418 (neomycin) (Gibco-BRL, Grand Island, N.Y.). Neo′ clones of K562cells were identified after 2 weeks, plucked, and maintained in RPMI/10%FCS containing 0.8 mg/mL neomycin. K562-neo′ cells cultured for 2 daysshowed a neo′ clonogenic cell doubling time of 36-42 hours.

Normal PBMCs (10⁴/mL) were spiked with 10% K562-neo′ cells, and NK-92cells were added to yield different effector:target (E:T) ratios ofNK-92:K562-neo′ cells. (Wong et al. (1996)). Briefly, PBMCs weresuspended in enriched alpha medium (Myelocult™) as described above. Thismedium has been shown to provide optimal conditions for supporting bothIL-2 activation of PBMCs and hematopoietic progenitor cell function(Klingemann et al., Exp. Hematol. 21:1263 (1993)). The finalconcentration of PBMCs in 35-mm tissue culture dishes (Corning, EastBrunswick, N.J.) was 1×10⁶/mL, and the proportion of input K562-neo′cells was kept at 10% for all experiments. Various numbers of irradiated(1000 cGy) NK-92 cells (see Examples 7 and 8) were added, resulting invarious E:T ratios as specified in Table 2. These mixtures were culturedin an atmosphere of 5% CO₂ in air for 4 or 48 hours at 37° C. with andwithout IL-2 (500 units/mL).

After the culture, cells were washed in RPMI/5% FCS, 10³ cells weresuspended in 0.8% Iscove's methylcellulose containing 0.8 mg/mLneomycin, and 1.1-mL volumes were plated in 3-mm petri dishes. After 7days at 37° C. in a humidified atmosphere of 5% CO₂ in air, colonieswere counted. The number of neo′ colonies provided a measure for thenumber of surviving clonogenic K562-neo′ cells present in the cellsuspension originally plated. Percent survival values for co-culturescontaining various numbers of NK-92 cells were determined by comparingthe number of clonogenic K562-neo′ cells present in test co-cultureswith the number of those present in control co-cultures (no NK-92 cellsadded) and harvesting after the same period of incubation. At an inputnumber of 10³ K562-neo′ cells prior to purging, the absolute number ofclonogenic K562-neo′ cells after 4 hours with no NK-92 cells present was6400±820 cells and after 48 hours 28,300±2100 cells. The mean±SEM offour to eight experiments is reported.

When only PBMCs were plated in the neomycin-containing methylcellulosemedium, no colonies were ever observed. To quantitate the purgingcapacity of NK-92 cells, PBMCs were spiked with 10% K562-neo′ cells andcultured for 4 or 48 hours in medium in the presence or absence of IL-2.Results, summarized in Table 2, show that NK-92 cells used at E:T ratiosof 10:1 and 5:1 eliminated the K562-neo′ cells from PBMCs, and that verylow survival was observed at E:T of 1:1. The presence of IL-2 during thepurging did not result in any increase in the number of K562 cellspurged compared to no IL-2 (Table 2).

TABLE 2 Purging effect of NK-92 cells % Survival (−IL-2) % Survival(+IL-2) NK-92:K562-neo^(r) of K562-neo^(r) of K562-neo^(r) E:T Ratio 4hrs. 48 hrs. 4 hrs. 48 hrs.  10:1 0 0 0 0   5:1 0 0 0 0   1:1 10.5 ± 2.115.4 ± 7.2  6.5 ± 3 15.2 ± 5.9  0.1:1   56 ± 14.1 68.5 ± 19.5 54.4 ± 1369.6 ± 16.8

Example 6. Effect of NK-92 Cells on Hematopoietic Progenitor Cells

The effect of NK-92 cells on PBMCs was determined (Cashman et al., Blood75:96 (1990)). Briefly, normal PBMCs were co-cultured with irradiated(1000 cGy) NK-92 cells (see Examples 7 and 8) for 2 days. Cells werethen plated in replicate 1.1-mL aliquots of methylcellulose-containingmedia at densities adjusted to give approximately 10-100 large coloniesof erythroid cells (from burst-forming units-erythroid [BFU-E]),granulocytes and macrophages (from colony-formingunits-granulocyte/macrophage [CFU-GM]), and combinations of all of these(from CFU granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]).Colonies were counted under an inverted microscope 2 weeks later.

The number and growth kinetics of clonogenic hematopoietic cells werequantified at a 1:1 ratio of NK-92:PBMC after 2 days of co-culture withirradiated (1000 cGy) NK-92 cells. The cells were plated in standardmethylcellulose and counted 2 weeks later. Results obtained from threedifferent normal donors are presented as percentage of normal controlsin Table 3. No growth inhibitory effect on hematopoietic progenitors byNK-92 cells was noted.

TABLE 3 Effect of NK-92 cells on colony formation of normalhematopoietic progenitor cells. Experiment Number CFU-GEMM BFU-E CFU-C 1100 46 94 2 200 98 64 3 33 104 103

Example 7. γ-Irradiation of NK-92 Cells

NK-92 cells were irradiated in T25 flasks (Corning, Newark, N.J.) withthe dose indicated using a cesium source (Cis-US, Bedford, Mass.). Adose range of 200-2000 cGy was tested. After irradiation, the cells werewashed twice in RPMI, resuspended in medium, and cultured for 72 hoursat 37° C. in the presence of 500 IU/mL IL-2. Cytotoxicity (⁵¹Cr-releaseassay) was performed with these cells as described above in Example 2.Prior to performing the ⁵¹Cr-release assay, the cells were left for 24hours in medium supplemented with IL-2 to allow for recovery.Proliferation was assessed by means of a ³H-thymidine incorporationassay. Prior to adding ³H-thymidine (0.5 μCi/cell), NK-92 cells wereresuspended in thymidine-free RPMI. Uptake of ³H-thymidine was measuredin a liquid scintillation counter 4 hours later. (Klingemann et al.,Leuk. Lymphoma 12:463 (1994)). The counts per minute (cpm) from threedifferent experiments are presented.

Clinical use of this cell line to purge cancerous cells requires thatNK-92 cells not undergo significant growth and proliferation. This wasachieved by irradiating the cells. Proliferation, as measured by ³Hincorporation, was effectively reduced at a dose of 1000 cGy (Table 4).The cytotoxicity of NK-92 cells after administration of variousradiation doses is presented in FIG. 3. At doses up to 1000 cGy anessentially undiminished cytolytic response was maintained.

TABLE 4 Effect of irradiation on the proliferation of NK-92 cellsExperiment Radiation dose (cGy) Number 0 500 1000 1500 2000 1 5766 3071 406  125 114 2 4236 2411 1216 1192 562 3 3894 2046  824  689 748

Example 8. Radiation Susceptibility of NK-92 Cells

NK-92 cells were irradiated by a γ ray source (Gammacell 40, AtomicEnergy of Canada. Ltd., Canada). A dose range of 100-3000 cGy wastested. After irradiation, the cells were washed and resuspended inculture medium with rhIL-2. Colony assays, viability and cytotoxicactivity of the irradiated NK-92 cells were performed using standardtechniques (Yan et al., Leukemia, 7:131-139 (1993)). To quantifyclonogenic NK-92 cells, NK-92 cells (500 cells per mL culture medium)were cultured in a 0.3% agar-based medium supplemented with 12.5% FCS,12.5% horse serum, 2 mM L-glutamine, 100 μg/mL penicillin 50 μg/mLstreptomycin, 10⁻⁵ M mercaptoethanol, and 500 U/mL rhIL-2 at 37° C. for14 days. An additional aliquot of 500 U/mL rhIL-2 was added at day 7during the culture. Triplicate cultures were performed for each datapoint.

The viability of NK-92 cells, determined by trypan blue staining, andthe recovery of the ability of the NK-92 cells to generate coloniesafter exposure to various radiation doses is shown in FIGS. 4 and 5,respectively. The NK-92 cells maintained substantial survival for 3 or 4days after exposure to high doses of radiation (1000-3000 cGy). However,in vitro clonogenic NK-92 cells were significantly depleted after lowdoses of radiation and totally eliminated by doses above 300 cGy. FIG. 6shows the cytotoxicity of NK-92 cells to K562, HL60 and 2patient-derived leukemic samples after exposure to the different dosesof radiation. Doses of 300, 500, and 1000 cGy allow for substantialcytolysis against leukemic cell lines and primary leukemias 1-2 daysafter radiation.

These experiments suggest that NK-92 cells, irradiated to an extent thatrenders them nonclonogenic, retain their cytolytic activity against awide spectrum of target cells. They therefore may be used ex vivo in thepurging of tumor cells as well as in the treatment of various cancers invivo.

Example 9. Cytolysis of Human Primary Leukemic Cells by NK-92

a. Patient-Derived Leukemic Samples.

Samples were obtained, with informed consent, during routine diagnosticblood studies or bone marrow (BM) aspirates from patients with newlydiagnosed or relapsed leukemias. 9 acute myeloid leukemia (AML) cases,11 chronic myeloid leukemia (CML) cases (6 chronic phase, 1 acceleratedphase and 4 blast crisis), 14 B-lineage-acute lymphoblastic leukemia(ALL) cases (13 pre-B-ALLs and 1 B-ALL) and 6 T-ALL cases, were studied(see Table 5). Blast-enriched mononuclear cells were isolated by FicollHypaque (Pharmacia, Piscataway, N.J.) density gradient separation andwashed in RPMI 1640 medium.

b. Effector Cells.

NK-92 cells were cultured and maintained in a-MEM medium supplementedwith 12.5% FCS, 12.5% horse serum and rhIL-2 (500 U/mL Chiron,Emeryville, Calif.). TALL-104 cells (a MHC-unrestricted human cytotoxicT cell clone, generously provided by Drs. D. Santoli and A. Cesano, TheWistar Institute, Philadelphia) were maintained in Iscove's modifiedDulbecco's medium supplemented with 10% FCS and rhIL-2 (100 U/mL)(Cesano et al., Blood, 87:393-403 (1996)). Another human NK cell clone,YT, was maintained in RPMI 1640 medium with 10% FCS and rhIL-2 (100U/mL) (Yodoi et al., J. Immunol., 134:1623-1630 (1985)).

c. Cytotoxicity Assays.

The cytotoxic activity of non-irradiated NK-92 and responding T cellsagainst leukemic targets was measured in a standard 4-hour chromiumrelease assay (CRA). Some of the samples were also measured in an18-hour CRA. A fixed number of ⁵¹Cr-labeled target cells (5×10³/well)was tested for susceptibility to 4 effector cell concentrations. ⁵¹Crrelease of target cells alone (spontaneous release, determined byplacing target cells in 5% Triton) was always <25% of maximal ⁵¹Crrelease. CRA data were expressed as specific lysis (%) at a giveneffector:target (E:T) ratio or were converted to lytic units (LU)defined as the number of effectors resulting in 30% lysis of targetcells (Cesano et al., Cancer Immunol. Immunother., 40:139-151 (1995)).The degree of sensitivity of patient-derived leukemic cell targets toeach effector was defined as insensitive (−/+: <10/10-19% lysis),sensitive (++/+++/++++: 20-29/30-39/40-49% lysis) and highly sensitive(+++++/++++++: 50-59/>60% lysis) at an E:T ratio of 9:1.

d. Results: Cytolysis of Human Primary Leukemic Cells by NK-92 Cells.

The sensitivity of patient-derived leukemic cells to the cytotoxiceffect of NK-92 cells is summarized in column 4 of Table 5. Of the 40patient-derived leukemic samples shown in Table 5, 26 (65%) weresensitive or highly sensitive to NK-92 mediated in vitro cytotoxicity.Six of the samples that were insensitive to the NK-92 cells in thestandard 4 hr CRA (sole or first entries), became sensitive after 18hours incubation (second entries, enclosed in parentheses). Leukemiablasts derived from 6 out of 9 (67%) AML, 6 of 6 (100%) T-ALL and 6 of14 (43%) B-lineage-ALL were either sensitive or highly sensitive to theNK-92 mediated lysis. 7 of 8 acute leukemia samples which demonstratedhigh sensitivity to the cytotoxic effect of NK-92 cells were derivedfrom relapsed patients and 1 was from a newly diagnosed patient. Out of11 CML samples, 8 (73%) were sensitive (5 in chronic phase) or highlysensitive (2 in blast crisis; 1 in accelerated phase) to the NK-92mediated cytolysis (Table 5).

In comparison, the last two columns in Table 5 present results obtainedwith cell lines known in the field to have cytolytic activity againsttumor cells, namely, TALL-104 cells and YT cells. Only 16 out of the 37leukemic samples tested (43%) were sensitive (4 AMLs, 5 B-lineage ALLsand 3 CMLs) or highly sensitive (1 AML, 1 B-lineage-ALL and 2 CMLs) tothe MHC unrestricted cytotoxic T cell clone TALL-104 mediated cytolysis.Leukemias sensitive to the TALL-104 cells were not consistentlysensitive to NK-92 cells, and cells that were lysed by NK-92 cells werenot always lysed by TALL-104 cells. In addition, the cytolytic activityof TALL-104 cells was usually detected only after 18 hours of incubation(second entries, enclosed in parentheses). Only four of 16 (25%) of thetarget samples that were lysed at 18 hours were also lysed in thestandard 4 hr CRA. The remaining 12 (75%) responded only after the 18hour incubation, with the response being generally lower than thatobserved with the NK-92 cells of the invention. Without wishing to bebound by theory, these observations may be due to the possibilitythat 1) different target structures are recognized by TALL-104 vs NK-92cells, or 2) a different pathway may be involved in the NK-92 and in theTALL-104 cell mediated cytolysis.

The majority of leukemic samples treated with YT cells, the otherNK-like clone tested, were found to be resistant, with the exception of2 samples (a CML in blast crisis and a T-ALL) (see Table 5).

In conclusion, the NK-92 cells of the invention are surprisingly andsignificantly more effective in lysing patient-derived tumor cells, andexert their effect in a shorter time, than do the cells from twocytolytic cell lines known in the field.

TABLE 5 Cytotoxicity of NK-92, T-ALL 104 and YT Clone to Patient-DerivedLeukemic Cells^(a) Disease Blast (%) in Cytotoxic Sensitivity PatientStatus Sample NK-92 TALL-104 YT AML 1 M4□ Relapse PB (66%) ++++++ +++++− 2 (M1) Relapse PB (50%) +++++ − − 3 (M3) Relapse PB (50%) +++ (++++) +(++++) − (−) 4 (M4) Refractory PB (90%) ++ (++) − (+) − (−) 5 (M2) NewBM (90%) +++ (+++) + (+++) ND 6 (M4) New BM (97%) − − − 7 (M4) New PB(39%) − (−) − (++) − (−) 8 (M3) New PB (55%) − (++) − (+++) + (−) 9 (M3)New BM (32%) − − − T-ALL 1 Relapse BM (98%) ++++++ − − 2 Relapse PB(85%) ++++++ − (−) +++ (+++) 3 Relapse PB (77%) ++++++ − (+) − (−) 4Relapse PB (60%) +++++ − (−) + (−) 5 New BM (40%) +++ − − 6 New BM (66%)+++ − − B-Lineage-ALL  1 ● Relapse BM (78%) +++++ ++++ −  2 New BM (30%)++++ ND ND  3 Relapse BM (75%) +++ (++++) + (++++) ++ (++)  4 New BM(97%) ++ (+++) + (+++) − (−)  5 Relapse BM (60%) + (+) − (+) − (−)  6Relapse BM (80%) − ND ND  7 Relapse PB (80%) − − (−) −  8 New BM (68%) −− −  9 New BM (33%) − − (+) − 10 Relapse BM (87%) − − (++) − 11 RelapseBM (75%) − (+++) − (++++) − 12 New BM (30%) − − ND 13 New PB (90%) −(+++) − (+++) ND 14 New BM (81%) − − ND CML  1 BC PB (45%) ++++++ ++++++++  2 AC PB (22%) ++++++ ++ −  3 BC PB (93%) +++++ ++ −  4 CP PB (15%)D++++ + −  5 CP PB (8%)D ++ (++++) ND ND  6 CP BM (12%)D + (+++) + (+) ND 7 CP BM (10%)D + (+++) + (++++) ND  8 BC PB (60%) + − −  9 BC BM(48%) + − (−) − 10 CP PB (21%)D + (++) − (++++) − (−) 11 CP PB (11%)D −− (+++++) − (−) Notes and Abbreviations. ^(a)) Columns show results ofchromium release assays at E:T = 9:1 after 4 h without parentheses, and(results after 18 h enclosed in parentheses); New: newly diagnosed; ND:none done; o: FAB classification; D: blast and promyelocyte; BM: bonemarrow; PB: peripheral blood; I: B-ALL; BC: blast crisis; AC:accelerated phase; CP: chronic phase.

Example 10. Cytotoxicity of NK-92 Towards Human Leukemic Cell Lines

The following human leukemic cell lines were cultured at 37° C. in 5%CO₂ in RPMI 1640 medium supplemented with 10% heat-inactivated fetalcalf serum (FCS), L-glutamine and antibiotics: K562 (Chronic myeloidleukemia in blast crisis), HL60 (acute promyelocytic leukemia), KG1(erythroleukemia), NALM6 (acute pre-B lymphoblastic leukemia), Raji(Burkitt's lymphoma), CEM/S (acute T lymphoblastic leukemic cell linesensitive to methotrexate (MTX), a commonly used antitumor drug) as wellas CEM/T (methotrexate-resistant subline of CEM/S) (Mini, E. et al.,Cancer Res. 45:325-330 (1985)).

NK-92 cells were highly cytotoxic to all the 8 leukemic cell linestested in a 4 hr standard CRA (Table 6). The MTX-sensitive T-ALL cellline CEM/S as well as its MTX-transport resistant subline CEM/Tdisplayed a similar sensitivity to the NK-92 cells. This suggests thattumors that are not responsive to MTX treatment could be treated byadministering NK-92 cells of the instant invention. Table 6 surprisinglyshows highly effective cytolytic activity for NK-92 against all thetarget cells tested. The results obtained at the low E:T ratio of 1:1are especially noteworthy.

In contrast, the cytolytic cell lines TALL-104 and YT have little orvirtually no cytolytic activity against many of these target cells underthese conditions; when active, their activity is generally lower thanthat for NK-92 at E:T of 1:1. TALL-104 was cytotoxic to K562, NALM6 andHL60 cells, however, Raji cells exhibited only 22.2% lysis at 9:1 E:Tratio and KG1 cells, CEM/S as well as CEM/T were resistant. The YT clonedid not exhibit significant cytotoxic activity. Activity was found onlyagainst K562 cells and Raji cells, which showed a 32% and 25% lysis at9:1 E:T ratio, respectively.

As shown in Table 6, NK-92 cells of the present invention have asignificantly wider range of action and higher activities than the knowncytolytic cell lines TALL-104 and YT. These activities are higher thanany previously reported values in the field of tumor cytotherapy.

TABLE 6 Specific Lysis of Human Leukemia Cell Lines by Natural KillerCell ClonesNK-92, TALL-104, and YT. Specific Lysis (%) NK92 TALL-104 YTEffector:Target Ratio Target 9:1 3:1 1:1 9:1 3:1 1:1 9:1 3:1 1:1 K56294.1 91.2 82.1 88.5 85.2 72.5 34.2 28.2 18.4 HL60 87.9 75.3 79.6 43.016.0 6.9 2.1 1.1 1.5 KG1 64.6 53.8 43.7 2.7 0.5 0 0.1 0 0 NALM- 72.656.8 52.4 67.8 55.6 33.3 1.0 0.5 0 6 Raji 86.0 75.4 70.0 22.2 10.2 0.325.1 18.0 14.2 TALL- 57.3 53.2 44.1 - - - 3.2 1.4 0.9 104 CEM/S 56.648.8 34.7 2.7 1.6 0.9 0.9 0.4 0.3 CEM/T 57.5 42.1 39.1 1.5 0.6 0.3 1.20.1 0.2

Example 11. Effect of NK-92 Cells on Normal Human Bone MarrowHematopoietic Cells

Heparinized bone marrow collected from normal donors was separated byFicoll Hypaque density gradient isolation to produce the mononuclearcells. Enrichment of hematopoietic cells and depletion of T cells wasachieved by soybean lectin agglutination (SLA) of mature marrow elementsand removal of residual T cells by resetting with sheep red blood cells(Reisner et al., Lancet, 2:327-31 (1981)).

Hematopoietic cell enriched fractions of normal bone marrows from 14normal donors were tested by standard CRA to determine theirsusceptibility to lysis by NK-92 cells. All of the normal bone marrowsamples were insensitive to NK-92 mediated cytolysis (FIG. 7).

Example 12. In Vivo Leukemogenesis of NK-92 Cells in SCID Mice

a. Experimental Animals.

Severe combined immunodeficient (SCID) mice (CB17 scid/scid andpfp/Rag-2) (6 to 8 weeks old; Taconic Farms, Germantown, N.Y.) weremaintained in microisolator cages under sterile conditions with aspecific pathogen-free environment. To determine the potential of NK-92cells to induce leukemia in vivo, 2×10′ viable NK-92 cells in 0.3 mLphosphate buffered saline (PBS) were administrated by eitherintraperitoneal (I.P.) or intravenous (I.V.) route every other day for 5injections in each animal. For subcutaneous (S.C.) inoculations, 2×10⁷NK-92 cells were injected in the right flank of SCID mouse, as describedpreviously (Yan et al., Blood, 88:3137-3146 (1996)). Thereafter, all theexperimental animals were administered rhIL-2, 5×10⁴ U every other dayfor 2 weeks by S.C. injection. Survival of the animals was followed forat least 6 months after inoculation.

b. Tissue Analysis.

From each group, 2 SCID mice were sacrificed at the end of observationand tissues from peripheral blood, bone marrow, spleen, liver, kidney,lung, and brain were collected for histopathological and/or fluorescenceactivated cell sorting (FACS) analysis. Tissue sections from sacrificedSCID mice were fixed in 10% neutral buffered formalin, dehydrated andembedded in paraffin, sectioned and stained according to standardhistological techniques.

Viable cells recovered from various tissues were stained by fluoresceinisothiocyanate-conjugated (FITC) or phycoerythrin-conjugated (PE) Mab,as described (Yan et al., (1996)). A FACS scan flow cytometer (BectonDickinson) was used for analysis. Monoclonal antibodies (Mabs) directedagainst the respective human cell surface antigens were used fordetermination of their presence: CD2, CD3, CD5, CD7, HLA-DR, CD45, CD56(Becton Dickinson). A fluorescein isothiocyanate (FITC)-conjugated ratanti-mouse Mab mCD45 (Boehringer-Mannheim, Indianapolis, Ind.) was usedfor characterization of murine leukocyte common antigen.

c. Leukemogenesis.

CB-17 scid/scid mice as well as pfp-Rag-2 mice were inoculated withNK-92 cells by I.V. (n=3, for each group), S.C. (n=2, each group) andI.P. (CB-17: n=8; pfp-Rag-2: n=3) injection. Survival of the animals wasfollowed at least 6 months after inoculation. At the end of the sixmonth period, all animals appeared healthy; there was nohepatosplenomegaly, lymphadenopathy or leukemic nodular growth, whichwould have indicated leukemia development. Leukemic cellularinfiltration was not detected in the different tissues of the sacrificedanimals by histopathology. No cells of human origin were detectable inthe tissues by FACS analysis.

Example 13. Comparison of Antileukemic Effect of NK-92 Cells with LAK,NK and T Cells Against Human Leukemic Cell Lines

To isolate the NK cell populations, a Ceprate^(R) cell separation systembased on avidin-biotin immunoaffinity (CeliPro, Bothel, Wash.) was usedto purify a CD56+ cell fraction from cultured LAK cells. Briefly, theharvested cells were washed and resuspended in PBS with 1% bovine serumalbumin (BSA). To each 1-2×10⁸ cells/mL, 40 μL primary monoclonalantibody (mouse anti-human CD56) was added and the cells were incubatedat 4° C. for 25 minutes. After incubation, the cells were washed andresuspended to a concentration of 1×10⁸ cells per mL in PBS with 1% BSA.Then, to each one mL cell suspension, 20 μL biotin labeled rat antimouseIgG1 antibody was added and the cells were incubated again at 4° C. for25 minutes. After incubation, the cells were washed and resuspended at aconcentration of 1×10⁸ cells per mL in PBS with 5% BSA and slowly passedthrough the avidin column. The CD56+ cells were captured; other cells,including the T cell fraction, were eliminated from the column. Afterwashing the column, the adherent cells were then disassociated from thecolumn by agitation and elimination. After separation, the NKcell-enriched populations contained >85% CD56+CD3− NK cells. Themajority of the other cells in the fraction (>95%) were CD3+CD56− Tcells.

To generate leukemia-reactive allocytotoxic T lymphocytes (CTLs),peripheral blood mononuclear cells (PBMC) isolated from normal donorswere cultured with irradiated leukemic stimulating target cells andirradiated autologous PBMC as feeder cells. Cultures were started in 60well plates at 1000 responder cells per well in RPMI 1640 mediumcontaining 15% human serum and rhIL-2 100 U/mL at 37° C., 5% CO₂. Theratios of stimulator cells and feeder cells to responder cells were 5:1and 10:1, respectively. After 10-12 days culture, CTLs were harvestedfrom growth-positive wells and specific lysis toward leukemic targetcells and K562 cells was quantitated by ⁵¹Cr-release assay. The CTLswere continuously cultured and fed with stimulator and feeder cells inflasks. After 2-3 weeks culture, the monoclonal antibody OKT3 (OrthoBiotech, Raritan, N.J.) was added to the culture for rapid expansion ofthe CTL lines.

The antileukemic effects of NK-92 cells, human LAK cells, NK cells(CD56+CD3−; CD56+ in FIG. 8), and T cells (CD3+CD56−; CD3+ in FIG. 8)were assessed by measuring in vitro cytolytic activity in standard CRA(FIG. 8, Panels A and B), and by measuring inhibition of leukemic cellxenograft growth in vivo (FIG. 8, Panels C and D) when the effectorcells and targets were co-inoculated subcutaneously into SCID mice. Inorder to evaluate the inhibition of growth in vivo, the area of thesubcutaneous growths of leukemic nodules as a measure of their size wasdetermined once a week after inoculation, and survival of the animalswas also followed. NK-92 cells displayed the highest in vitrocytotoxicity against K562 (FIG. 8, Panel A) and HL60 (FIG. 8, Panel B)of the cells tested, with a mean specific lysis of 89% and 78%,respectively. This was superior to the killing mediated by human LAK(52% and 11%, respectively), NK (72% and 28%, respectively) and T cells(12% and 1.2%, respectively).

Correspondingly, the NK-92 cells demonstrated more effective in vivoinhibition of the growth of K562 (FIG. 8, Panel C) and HL60 (FIG. 8,Panel D) leukemic cells xenografts than did the human LAK and NK cells.The results shown in FIG. 8 indicate that the NK-92 cells of the presentinvention have cytolytic activity in vitro and tumor-inhibiting activityin vivo that is superior to those activities manifested by the knownpreparations of cytolytic cells normally present in humans. Theseactivities are therefore unexpected by a worker in the field of tumorcytotherapy.

Example 14. Comparison of Antileukemic Effect of NK-92 Cells withAllogeneic Leukemic-Reactive CTL Cells

To examine the in vivo effects of NK-92 cells and other effector cellson the growth of human leukemia xenografts, 5×10⁶ leukemic target cellsalone or mixed with 2×10⁷ NK-92 or other effector cells (E:T ratio=4:1)were injected S.C. into SCID mice. The TALL-104 effector cells wereirradiated with 3000 cGy before inoculation to prevent leukemogenesis inSCID mice. RhIL-2 was administered to the mice, as in Example 13. TheLog-rank test and Wilcoxon test were used for the comparison of thesurvival of leukemia bearing SCID mice.

The antileukemia effect of NK-92 cells was evaluated using allogeneicleukemia-reactive CTL cells (derived from a patient with T-ALL (TA27)).Both NK-92 and CTL cells activated by exposure to TA27 displayed asignificantly higher specific cytolysis (70% and 58% at 9:1 E:T ratio,respectively) than the other effectors (LAK cells: 22%; NK cells(designated CD56+ in FIG. 9): 38%; TALL-104: 8%; and T cells (CD3+ inFIG. 9): 1.5% specific lysis) against the TA27 leukemic cells (FIG. 9,Panel A). Correspondingly, the subcutaneous growth of TA27 leukemiccells was inhibited by co-injection of either NK-92 cells oranti-TA27-CTL cells (FIG. 9, Panel B). The survival of those animalswhich were co-inoculated with TA27 leukemic cells plus NK-92 or withanti-TA27-CTL cells was significantly prolonged beyond that of theanimals bearing TA27 leukemia alone (NK-92 cells: p=0.001; TA27-CTLcells: p=0.002; see FIG. 10). In contrast the TALL-104 cells did notshow significant in vitro killing against TA27 leukemic cells by CRA(FIG. 9, Panel A). However, moderate inhibition of the leukemic tumorgrowth in vivo (FIG. 9, Panel B), coupled with a statisticallyinsignificant (p>0.05) increase in survival, was observed in the animalsco-inoculated with TA27 leukemic cells and irradiated TALL-104 cells(FIG. 10).

Example 15. Antileukemia Effect of NK-92 Cells in Human LeukemiaXenograft SCID Mice Model

For study of the in vivo tumoricidal capacity of NK-92 cells, leukemiccells derived from a T-ALL patient (TA27), an AML patient (MA26), and apre-B-ALL patient (BA31) were adoptively grown and expanded in SCID miceby S.C. inoculation. Leukemic cells recovered from the leukemic nodulesin the mice (first passage) were used in these experiments. The SCIDmice in each group were inoculated I.P. with 5×10⁶ leukemic cells fromthe first passage in 0.2 mL PBS, and 24 hours later 2×10⁷ NK-92 cells in0.4 mL PBS were administered by I.P. injection. The animals receivedeither 1 dose or a series of 5 doses of NK-92 cells which wereadministered on days 1, 3, 5, 7, and 9, with and without rhIL-2, asindicated in the Figures.

All the human leukemias grew aggressively in SCID mice. Leukemic cellsderived from a patient (TA27) with T-ALL and a patient (MA26) with AMLM4 leukemia were highly sensitive in vitro to the NK-92 cells (73% and66% specific killing at 9:1 E:T ratio determined by CRA, respectively),whereas cells from a patient with pre-B-ALL (BA31) were insensitive tothe NK-92 cells (4% specific killing at 9:1 E:T ratio assessed by CRA).FIG. 11 shows that the survival of mice bearing TA27 leukemia wassignificantly prolonged by the administration of NK-92 cells. The mediansurvival time (MST) of the animals with no treatment or rhIL-2 alone was72 days (n=5) and 63 days (n=5) (p>0.05), respectively. All theseanimals died of leukemia. Treatment with NK-92 cells (alone or withrhIL-2) increased the MST to 102 days (n⁼6) and 114 days (n=6),respectively, for the 1 dose injection schedule (2×10⁷ NK-92 cells, day1). The MST increased to 160 days (n=6) and 129 days (n=6),respectively, with 5 doses NK-92 with or without rhIL-2 injection (FIG.11). Three animals that received 5 doses of NK-92 cell injections withor without rhIL-2 administration survived without any signs of leukemiadevelopment 6 months after inoculation. There was no significantdifference in survival between the mice receiving treatments with orwithout rhIL-2 administration, whether in the group receiving 1 dose ofNK-92 cells (p=0.75), or the in the group receiving 5 doses (p=0.45).Compared to the group receiving 1 dose of NK-92 cells, with or withoutrhIL-2 treatment, survival was significantly extended in animals thatreceived 5 doses of NK-92 cells without rhIL-2 treatment (p=0.009 andp=0.009, respectively).

In SCID mice inoculated with human pre-B-ALL (BA31) leukemia, with orwithout rhIL-2 treatment, the MST were 63 days (n=5) and 64 days (n=5),respectively (see FIG. 12). For the animals that received 5 doses of2×10⁷ NK-92 cells, with or without rhIL-2 administration, the MST wasincreased to 79 days (n=5) and 76 days (n=5), respectively. Thesesurvival times were not significantly different from those for theanimals that were not treated by NK-92 cells (p>0.05).

In animals bearing human AML (MA26), MST was 97 days (n=6) (see FIG.13). The MST was extended to 173 days among the animals that received 5doses of 2×10⁷ NK-92 cells (p<0.01)(n=6). Three of the 6 animals thatreceived NK-92 cells remained alive 6 months after leukemia inoculation.Two of these appeared healthy without any signs of leukemia development.One mouse had an enlarged abdomen indicating residual leukemia. The 6animals that received NK-92 cells plus rhIL-2 treatment were all alive 6months after leukemia inoculation without any signs suggestive ofleukemia development.

The results presented in FIGS. 11-13 show that in vivo treatment ofleukemic tumors can result in enhanced longevity of the subject mice.The extent of the prolongation of life, and of the improvement in thehealth of the animals, is dependent on the particular leukemic tumorinvolved, and ranges from modest or insignificant (FIG. 12) to verydramatic (FIG. 13). Based on these results, it is concluded thattreatment of tumors in vivo by administering NK-92 cells, depending onthe tumor in question, can be surprisingly effective.

Example 16. Preparation of Modified NK-92 Cell Lines Secreting IL-2

In order to generate NK-92 cells that constitutively secrete IL-2, twoplasmids encoding human IL-2 were employed.

a. Methods.

DNA Clones: The MFG-hIL-2 vector (FIG. 14) was generously provided byDr. Craig Jordan (formerly of Somatix Corp., Alameda, Calif.). ThepCEP4-LTR-hIL-2 vector (FIG. 15) was created by excising the HinDIII-Barm HI fragment from the MFG-hIL-2 vector, containing the 5′ LTRand hIL-2 gene, and inserting it into the complementary sites of thepCEP4 episomal vector backbone (InVitrogen, Carlsbad, Calif.).

Particle-Mediated Gene Transfer: NK cells were transduced byparticle-mediated gene transfer using the Biolistic PDS-1000/He ParticleDelivery System (BioRad Laboratories, Hercules, Calif.). Cells weretransduced according to the manufacturer's instructions. Briefly, 1.0 or1.6 μm gold particles were coated with 5 μg of DNA using calciumchloride spermidine, and ethanol. NK-92 cells were prepared forbombardment by adherence to poly-L-lysine (Sigma, St. Louis, Mo.) coated35 mm tissue culture plates. Cells were bombarded in an evacuatedchamber (vacuum of 20 inches mercury) and DNA-coated particles wereaccelerated by a 1,100 psi helium pulse. Cells were returned to IL-2supplemented Myelocult media immediately following bombardment andallowed to recover for 24 hours prior to transfer to IL-2-free media.Media was changed periodically. Cells were selected for IL-2-independentgrowth. Preliminary experiments showed heat transfer efficiencies of5-15% were obtained under the conditions used.

PCR and Southern Blot Analysis: The transfection of the NK-92 cells wasconfirmed by polymerase chain reaction (PCR) analysis of DNA isolatedfrom both the parental and transfected NK-92 cell lines for the presenceof genomic and cDNA forms of the human IL-2 gene. DNA was isolated usingDNAzoI (Gibco Life Technologies Inc., Burlington, ON). Briefly, cellswere lysed in DNAzoI and DNA was precipitated with ethanol at roomtemperature. DNA pellets were collected, washed in 95% ethanol andbriefly air dried. DNA was resuspended in 8 mM NaOH at 62° C. and thesolution was neutralized with HEPES buffer. DNA was quantitated byabsorbance at 260 nm. Primers flanking exon 1 of the human IL-2 gene(forward: 5′-CAA CTC CTG TCT TGC ATT GC-3′ (SEQ ID NO:1) and reverse:5′-GCA TCC TGG TGA GTT TGG G-3′ (SEQ ID NO:2), Gibco Lift TechnologiesInc., Burlington, ON) were used to amplify the DNA (30 cycles, 1 min 95°C., 2 min 50° C. and 2 min 72° C.). PCR products were resolved on a 2%agarose gel. For Southern blot analysis, DNA was transferred toHybond+nylon membrane (Amersham Life Sciences, Arlington Heights, Ill.)by capillary transfer in 10×SSC (1.5M NaCl, 1.5M NaCitrate) and fixed byUV cross-linking (StrataLinker Stratagene, La Jolla, Calif.). The blotwas hybridized with a ^(32P) radiolabeled human IL-2 probe for 8-12hours, washed and visualized by autoradiography at −70° C. with KodakX-Omat XAR film.

Northern Blot Analysis: Cytokine and chemokine gene expression wasanalyzed by Northern blot analysis. RNA was extracted from parental andtransfected NK-92 cell lines using Trizol reagent (Gibco LifeTechnologies Inc., Burlington, ON) according to the manufacturer'sinstructions. Briefly, cells were lysed in Trizol and the lysateextracted with chloroform. The aqueous phase was then precipitated withisopropanol. The RNA pellet was collected, briefly air-dried and thenresuspended in DEPC-treated water (diethyl-pyrocarbonate; Sigma ChemicalCo., St. Louis, Mo.). RNA was quantitated by determining OD_(D). Fifteenmicrograms of RNA was resolved on a 1% formaldehyde agarose gel in 1%MOPS (3-[N-Morpholino]propanesulfonic acid, Sigma, St. Louis, Mo.) andblotted as described previously for Southern blot analysis. The blot washybridized with ³²P radiolabeled probes for human IL-2 and TNF-α.

DNA probes for Northern and Southern blot analysis were radiolabeled byrandom primer extension. DNA probes for human IL-2 and TNF-α werepurified by digestion with appropriate restriction endonucleases andagarose electrophoresis. The DNA was excised from the gel and purifiedby centrifugation through a Spin-X tube filter (Corning Costar,Cambridge, Mass.), phenol:chloroform extraction and ethanolprecipitation. DNA probe was labeled with a ³²P-dCTP (Sp. Ac. 3000Ci/mmol; ICN, Montreal, PQ).

Cytokine Determination: IL-2 production by NK-92 cell lines wasdetermined by ELISA. Aliquots of 1×10⁶ of the parental or transfectedNK-92 cells were cultured in 8 ml of IL-2 free Myelocult media for 1, 2,and 3 days. Supernatants were collected from at −20° C. until allsamples were collected. Samples were thawed and assayed for IL-2 levelsby ELISA according to the manufacturers' instructions (Quantikine; R&DSystems, Minneapolis, Minn.). The ELISA is a horseradishperoxidase/tetramethylbenzidine based colorimetric assay and the ELISAmicrotiter plates were read at 450 nm (with a 540 nm correction) in amicroplate reader (Model EK309, Bio-Tek Instruments Inc., Winooski,Vt.).

Irradiation of NK-92 Cells: To determine the sensitivity of bothparental and transfected NK-92 cells to irradiation, cells wereirradiated using a Cis BioInternational 437c cesium source (Cis-US,Bedford, Mass.). Cells were collected, washed and resuspended in mediumand irradiated in 15 or 50 ml conical centrifuge tubes (BectonDickinson, Franklin Lakes, N.J.). Following irradiation, cells werewashed and resuspended in Myelocult with (for parental NK-92) or without(for transfected cells) IL-2. Cells were cultured for 24, 48 and 72hours and assayed for viability by trypan blue exclusion, forproliferation by ³H thymidine incorporation and for cytotoxicity by⁵¹Cr-release assay (as described above).

b. Plasmid MFG-hIL-2.

For NK cells transfected with the MFG-hIL-2 vector, 85-95% of cells diedafter 4-7 days following transfer to unsupplemented media. A smallnumber of cells, however, remained viable. These were assumed to becells that had been successfully transfected. However, even with thesecells, no viable cells were detectable after two to three weeks. Thiswas expected as the MFG-hIL-2 vector construct did not contain thegenetic elements required for replication and maintenance in eukaryoticcells such as a mammalian origin of replication. Therefore, as thetransfected cells were maintained in culture and began to replicate, thevector construct would have been lost from cells and the cells wouldhave reverted to their IL-2-dependent phenotype. These cultures werenevertheless propagated for several weeks. Surprisingly, a small numberof viable cells appeared in the cultures after approximately 4-5 weeksfollowing initial transfer of the cells to IL-2-free media. These cellswere capable of IL-2-independent growth upon subculturing to fresh mediaand appeared to be stably transfected, maintaining their IL-2independent phenotype during prolonged culturing. Since the vector wasunable to replicate, the appearance of stably transfected cells suggeststhat the vector had integrated into the genome of a transfected cell.Since this would be a very rare event, these transfected cells probablyarose from one or a very small number of cells. IL-2-independent NK-92cells arising from transfection with the MFG-hIL-2 were denoted asNK-92MI.

c. Plasmid pCEP4-LTR.hIL-2.

Initial observations for cells transfected with the episomal vectorpCEP4-LTR.hIL-2 were identical to those seen with NK-92MI. The majorityof the transfected cells died within 4-7 days following transfer toIL-2-free Myelocult media. However, unlike the NK-92MI cells, theremainder of the cells did not lose their IL-2-independent phenotype orvitality and die after the initial 2-3 week period. Instead, the cellsthat were initially IL-2-independent were immediately capable oflong-term IL-2-independent growth and survival. This was expected sincethe pCEP-LTR.hIL-2 vector contains elements that enable it to bemaintained in eukaryotic cells as an autonomously replicating geneticelement. Therefore, any cell that was initially transfected shouldmaintain its IL-2-independent phenotype for an indefinite length oftime. Although cells harboring episomal vectors are not stablytransferred by strict definition, these cells are under constantselection pressure in IL-2-free media in favor of cells maintaining thevector. Therefore, these cells are capable of long-term culturing:IL-2-independent NK-92 cells arising from transfection with thepCEP4-LTR.hIL-2 are denoted as NK-92CI.

To confirm that NK-92MI and NK-92CI had in fact been transfected withhIL-2 gene, PCR analysis was performed on the parental and transfectedcell lines. Primers flanking exon 1 of the hIL-2 gene, which has 88 basepairs (bp), were used to amplify DNA isolated from NK-92, NK-92MI andNK-92C1 to assay for the presence of the genomic and cDNA forms. Agarosegel electrophoresis of the PCR products from the parental line revealeda single 263 bp fragment corresponding to the size expected for the DNAfragment amplified from the genomic IL-2 gene. However, analysis of boththe NK-92MI and NK-92CI products revealed two bands, the 263 bp fragmentcorresponding to the genomic hIL-2 gene as well as a 175 bp fragmentresulting from the amplification of the hIL-2 cDNA. To confirm theidentity of these DNA fragments, Southern blot analysis with aradiolabeled probe specific for hIL-2 was performed. Both the 263 bpgenomic fragment and the 175 bp cDNA fragment hybridized with the probe.These data indicate that both NK-92MI and NK-92CI had been successfullytransfected and contained the cDNA for hIL-2.

d. Analysis of Gene Expression.

Expression of specific cytokines in the parental and transfected celllines, they were analyzed by Northern blot analysis. RNA isolated fromNK-92, NK-92MI, and NK-92CI cells was separated by electrophoresis,transferred to a nylon membrane and hybridized with probes for thecytokines hIL-2 and hTNF-α. Northern blot analysis of IL-2 revealed thatIL-2 RNA was not detectable in the parental cell line. However, hIL-2RNA was detected in both NK-92MI and NK-92CI. Two mRNA transcripts weredetected in NK-92MI cells: a major RNA species of approximately 1.9 kDaand a less intense transcript at 2.4 kDa. In NK-92CI, a hIL-2 mRNAtranscript of approximately 1.4 kDa was detected. A very faint band wasalso seen at 2.5 kDa. These data confirm that the transfected cellsexpressed IL-2 while the parental NK-92 cells did not. The significanceof the multiple hIL-2 mRNA transcripts in the two transfectants is notclear, although it is possibly a consequence of the different vectorconstructs. Furthermore, in the case of NK-92MI, the integration of thehIL-2 gene into the genomic DNA may also have affected the RNA size.

TNF-α expression in the NK cells was also examined using this technique.It was seen that all three lines expressed the gene for this cytokine. ATNF-α probe hybridized to a 1.6 kDa band in RNA isolated from NK-92,NK-92MI and NK-92CI. These results indicate that although transfectionof NK-92 cells with the IL-2 gene resulted in expression of the IL-2 inthe transfectants, this did not influence the expression of anothercytokine.

e. Secretion of hIL-2.

After confirming expression of the IL-2 gene by Northern blot analysis,cells were assayed for production and secretion of hIL-2 by ELISA.Aliquots of 10⁶ NK-92, NK-92MI and NK-92CI cells were plated in 8 mLaliquots and cultured in Myelocult in the absence of IL-2. Supernatantswere collected after 24, 48 and 72 hours for IL-2 analysis by ELISA.Background levels of IL-2 were detected in the supernatant of NK-92cells at all three time points (2-3 pg/mL). Elevated IL-2 levels weredetected in both NK-92MI and NK-92CI supernatants (Table 7). NK-92MIproduced much higher levels of IL-2 in comparison to NK-92CI, withlevels ranging from 60× higher after 24 hours (9.3 pg/mL vs 549.3 pg/mL)to about 80× higher after 48 hours (15.7 pg/mL vs 1,260.3 pg/mL) and 72hours (27.2 pg/L vs 2,248.3 pg/mL).

TABLE 7 Synthesis of Human IL-2 by NK-92, NK-92MI, and NK-92CI IL-2(pg/ml) in Experiment #1 #2 #3 Ave ± S.D. NK-92 Day 1 0 7 1   2.7 ± 3.8Day 2 0 4 1   1.7 ± 2.1 Day 3 0 3 3   2.0 ± 1.7 NK- Day 1 517 568 545 549.3 ± 34.7 92Ml Day 2 977 1462 1342 1260.3 ± 252.6 Day 3 1872 26102263 1148.3 ± 369.2 NK- Day 1 7 13 8   9.3 ± 3.2 92Cl Day 2 14 16 17 15.7 ± 1.5 Day 3 52 18 13  27.7 ± 21.2

f. Comparison of Cell Surface Antigens in NK-92, NK-92MI and NK-92CI.

To compare the IL-2-independent transfectants with the parental cells,NK-92MI and NK-92CI were analyzed for CD2, CD3, CD4, CD8, CD10, CD16,CD28, CD56, ICAM-1, ICAM-2, ICAM-3 and LFA-1 expression by fluorescentactivated cell sorting (FACS) analysis. The transfected cells revealed apattern of expression identical to that seen on the untransfectedparental cell line with the exception of the IL-2 receptor. FACSanalysis of CD25 (the IL-2 receptor α-chain) on NK-92 cells indicatedthat the receptor was expressed on the surface of NK-92 cells and thatits expression is down-regulated in response to IL-2. This confirmedsimilar findings obtained in earlier work (Gong et al., 1994).Therefore, NK-92 cells in unsupplemented media had relatively highlevels of CD25 on their surface while cells in media supplemented withas low as 100 U/mL had low levels of CD25 cell surface expression.

CD25 expression in the high IL-2-producing transfectant NK-92MI wasdecreased both in unsupplemented media and in media supplemented with100 U/mL or 1000 U/mL of IL-2. These results are consistent with thoseseen with the parental cells. Since the levels of endogenously producedIL-2 in NK-92MI were high, down-regulation of IL-2 receptor levels isexpected even in the absence of exogenously administered IL-2.

Culture of NK-92CI in media supplemented with 100 U/mL and 1000 U/mLIL-2 resulted in CD25 upregulation and increased cell surfaceexpression. However, the results for NK-92CI in unsupplemented media arenot as clear. Two distinct populations appear, a population expressingvery low CD25 levels, similar to NK-92MI, and a population expressinghigh levels, similar to the NK-92 parental cells. This suggests thatNK-92CI consists of a polyclonal population consisting of high and lowIL-2 expressing cells rather than a uniform population of cellsexpressing an intermediate to low level of IL-2. Therefore, whencultured in IL-2-free media, the cells expressing high levels of IL-2would have low surface levels of CD25 while low IL-2 expressing cellswould have high CD25 levels on their surface.

Example 17. Cytotoxicity of NK-92 Transfected to Produce IL-2

To evaluate the cytotoxicity of these transfected cells, a standard 4hour ⁵¹Cr-release assay was performed to compare the toxicity of theparental cells to NK-92MI and NK-92CI to the standard test target cellsK562 and Raji. The cytotoxicity of NK-92MI and NK-92CI was comparable tothat seen with the parent cells (FIG. 16). The transfected cell linesshow cytotoxic activities against K562 and Raji that are very similar tothat of the parental cells. Cytotoxicity of NK-92 against K562 rangedfrom 82 to 67% while NK-92MI and NK-92CI had cytotoxicity ranges of 77to 62% and 82 to 62%, respectively. For Raji cells, NK-92 hadcytotoxicity of 81 to 47%, NK-92MI had cytotoxicity of 75 to 65% andNK-92CI had cytotoxicity of 82 to 52%.

Example 18. Effect of Transfected NK-92 Cells on HematopoieticProgenitor Cells

One potential clinical application of the NK-92, NK-92MI and NK-92CIcells is as an ex vivo purging agent for autologous grafts. In order forthe NK cells to be suitable for such a purpose, they must be able topurge the malignant cells without killing the hematopoietic progenitorcells in the graft or influencing their hematopoietic potential. Inorder to assay this, a colony-forming cell assay (CFC) was performedwhere the clonogenic output of PBMCs was examined following a 48 hourincubation with NK-92MI and NK-92CI at various E:T ratios. NK-92 waspreviously shown to have minimal effect on hematopoietic stem cells(Example 6). In this example, NK-92MI and NK-92CI also show little or noeffect on clonogenic output. The number of total colonies followingincubation with either NK-92MI or NK-92CI was very similar to control,although a slight decrease was seen with the highest effector:PBMC ratioof 1:1 (FIG. 17). Total clonogenic output from both NK-92MI and NK-92CIwas approximately 80% of control under this condition. However, noconsistent trend was seen in terms of clonogenic output with respect tothe ratio of NK:PBMCs. In terms of specific colony types, there were nodetectable differences in the number of output BFU-E colonies, which arethe most numerous. Some effect was seen with both the CFU-GM andCFU-GEMM colonies. However, the absolute numbers of these colonies arevery low, making any conclusions difficult since small variations in thenumber of colonies has a large effect on the calculation of clonogenicoutput. An influence on CFU-GM and CFU-GEMM is seen at higher ratios,but no consistent correlation between ratio and output was noted.

Example 19. Irradiation of the Transfected NK-92 Cells

To establish an effective irradiation dose to inhibit proliferation andmaintain cytotoxicity, NK-92MI and NK-92CI cells were irradiated at 500,1,000, 1,500 and 2,000 cGy and assayed for proliferation by the ³Hthymidine incorporation assay (see Examples 7 and 8). Both NK-92MI andNK-92CI were more sensitive to irradiation than the parental NK-92 cell.Proliferation of NK-92MI and NK-92CI was found to be more stronglysuppressed than NK-92 at all radiation doses tested (FIG. 18, Panel A).For NK-92MI and NK-92CI, proliferation was completely suppressed by aradiation dose between 500 and 1,000 cGy. The level of thymidineincorporation reached a plateau at approximately 20% of unirradiatedcontrol cells for NK-92CI and 10% for NK-92MI. For determination ofviability, NK-92, NK-92MI and NK-92CI cells were irradiated at 250, 500,1,000 and 2,000 cGy and trypan blue exclusion was determined 24, 48 and72 hours following irradiation. It was found that greater percentages ofboth NK-92MI and NK-92CI were found to be killed by irradiation ascompared to the parental cells at equivalent doses (FIG. 18, Panel B).Viability of NK-92 was higher than that of both transfectants at alldose rates tested.

The cytotoxicity of these cells following irradiation is shown in FIG.19. Cells irradiated at 0, 1,000 and 2,000 cGy were tested after threedays for cytotoxicity against K562 and Raji cells at effector:targetratios of 20:1, 10:1, 5:1 and 1:1. Cytotoxicity of NK-92 cells threedays following irradiation at 1,000 cGy was determined to beapproximately 10-30% K562 (FIG. 19, Panel A) and 30-50% and for Raji(FIG. 19, Panel B). Irradiation at 2,000 cGy resulted in cytotoxicity of1-5% against K562 and 3-13% against Raji. In contrast, NK-92MI had only0-5% and 0-1% cytotoxic activity against K562 and 0-1% and 0% againstRaji three days after irradiation doses of 1,000 and 2,000 cGy,respectively. NK-92CI had only 1-4% cytotoxicity to K562 and 2-7% toRaji three days after irradiation at 1000 cGy and 0% to K562 and 0-2%after irradiation with 2000 cGy.

In the data reported here, IL-2 transfectants are seen to be moresensitive to irradiation than the parental strain. Proliferation andcytotoxicity of both NK-92MI and NK-92CI cells were suppressed at alower radiation level than for the parental strains, andradiation-induced lethality was much greater in the IL-2-independentmodified cells in NK-92 at equivalent radiation doses. The highIL-2-producing NK-92MI is more sensitive to radiation than the low IL-2producing NK-9201 variant. As a result of the increased radiationsensitivity, a reduced level of irradiation would be sufficient toadequately control proliferation while minimizing lethality to the cellsand inhibition of cytotoxicity. In routine experiments, the worker ofordinary skill would be able to repeat experiments such as thosedescribed in this example. By using lower radiation doses, in the rangebetween 0 and 1000 cGy optimal doses can be determined that inhibitproliferation while maintaining viability and cytolytic activity inNK-92MI and NK-92C1.

Example 20. Transfection of NK-92 with a Gene for Thymidine Kinase

NK-92 cells are to be transfected with a vector bearing a gene forthymidine kinase (TK). The resulting TK-modified NK-92 cells are therebyrendered susceptible to the toxic effects of the guanosine analogs,gancyclovir, and acyclovir.

A vector suitable for transfecting a mammalian cell is to beconstructed, such as a retroviral vector harboring a herpes simplexvirus (HSV) TK gene, under the control of the HSV TK promoter, andcontaining its own polyA addition site. Transfection is to be carriedout by a method known to those skilled in cell biology and mammalianmolecular biology, such as by electroporation (Bio-Rad Gene Pulser™), orby lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413(1987)). The transfected NK-92 cells so produced are susceptible toinactivation by administering gancyclovir or acyclovir.

Example 21. Mutation of NK-92 HLA Cell Surface Protein

NK-92 cells are to be obtained from the cell line described by Gong etal. (1994). The chromosome bearing the β₂-microglobulin gene is to beisolated, and the DNA contained within this chromosome is to be purifiedaway from histones and other DNA-bound proteins. The gene fragmentbearing β₂-microglobulin is to be excised with restriction nucleases,and site specific mutagenesis is to be conducted via an oligonucleotidecassette harboring the mutated nucleotide sequence. These proceduresemploy techniques commonly known in recombinant DNA technology, as setforth, for example, in “Current Protocols in Molecular Biology”, Ausubelet al., John Wiley and Sons, New York 1987 (updated quarterly), and“Molecular Cloning: A Laboratory Manual”, 2nd Ed., Sambrook, Fritsch andManiatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989,incorporated herein by reference. The mutated β₂-microglobulin is to bereincorporated into the cellular DNA, and reintroduced into the NK-92cells. This preparation of cells will then express cell surface HLAmolecules incorporating mutated β₂-microglobulin moieties, and will havelost the ability to bind T-cell receptors.

Example 22. NK-92 Cells Expressing Receptors for a Cancer Cell

The CTL of a patient suffering from a cancer are to be harvested bydifferential centrifugation on a density gradient. The CTL are to beimmunoaffinity purified to contain predominantly the CTL targeting areceptor on the cancer cell from the patient. The DNA of the CTLpopulation obtained is to be isolated, and the genes for the MHC class Ireceptor in the cancer-targeted CTL isolated by restriction nucleasecleavage. The genes so purified are to be amplified using the polymerasechain reaction, and the resulting amplified genes incorporated into avector suitable for the constitutive expression of the genes in NK-92cells. The vectors are to be transfected into NK-92 cells, and themodified NK-92 cells so obtained are to be selected using, for example,an antibiotic resistance marker incorporated into the vector. The cellsso selected are to be cultured to increase their number. They may thenbe employed to target specifically the cancer cells in the patient, andtreat the cancer occurring in the patient either ex vivo or in vivo.

Example 23. Use of NK-92 Cells to Kill HIV-Infected Cells

8E5 is a cell line harboring HIV that produces HIV virions. 8E5L is acorresponding cell line infected with HIV which does not producevirions. In a cytotoxic activity experiment in which the chromiumrelease assay was used to evaluate activity, the results presented inTable 8 were obtained. In these experiments, A3.01 cells are anuninfected control cell line.

TABLE 8 Cytotoxic Activity of NK-92 Cells on HIV-Infected Cells. TargetE:T Ratio % Cytotoxicity A3.01 50:1 43 20:1 51  5:1 44  1:1 44 8E5L 50:143 20:1 37  5:1 44  1:1 40 8E5 50:1 76 20:1 69  5:1 77  1:1 65

It is seen from Table 8 that 8E5 cells which produce HIV particleselicit a higher cytotoxic activity than do 8E5L cells, which do notproduce HIV particles, and higher than control cells. Without wishing tobe bound by theory, it is believed that the anti-viral effect of NK-92cells is due to factors such as a direct cytotoxic effect, as well asinhibition through MIP-1α, which is produced by NK-92 cells in highconcentrations (Bluman et al, J. Clin. Investig. 97, 2722 (1996)). Theresults indicate that NK-92 cells effectively lyse HIV-producing cellsin vitro.

I claim:
 1. A method of treating a cancer in vivo in a mammal comprisingthe step of administering to the mammal a medium comprising an NK-92cell line ATCC Deposit No. CRL-2407, wherein said cancer is recognizedand lysed by said NK-92 cell line and wherein said cancer is a solidtumor.